Antibody-drug-conjugate and its use for the treatment of cancer

ABSTRACT

The present invention relates to an antibody-drug-conjugate. From one aspect, the invention relates to an antibody-drug-conjugate comprising an antibody capable of binding to a Target, said antibody being conjugated to at least one drug selected from derivatives of dolastatin 10 and auristatins. The invention also comprises method of treatment and the use of said antibody-drug-conjugate for the treatment of cancer.

The present invention relates to an antibody-drug-conjugate. From oneaspect, the invention relates to an antibody-drug-conjugate comprisingan antibody capable of binding to a target, said antibody beingconjugated to at least one drug selected from derivatives of dolastatin10 and auristatins. The invention also comprises method of treatment andthe use of said antibody-drug-conjugate for the treatment of cancer.

BACKGROUND OF THE INVENTION

The invention relates to an antibody-drug-conjugate (ADC) or conjugateand its use for the treatment of cancer.

ADCs combine the binding specificity of an antibody with the potency ofdrugs such as, for example, cytotoxic agents. The technology associatedwith the development of monoclonal antibodies, the use of more effectivedrugs and the design of chemical linkers to covalently bind thesecomponents, has progressed rapidly in recent years.

The use of ADCs allows the local delivery of drugs which, ifadministered as unconjugated drugs, may result in unacceptable levels oftoxicity to normal cells.

In other words, maximal efficacy with minimal toxicity is soughtthereby. Efforts to design and refine ADC have focused on theselectivity of antibody as well as drug mechanism of action,drug-linking, drug/antibody ratio (loading or DAR), and drug-releasingproperties. Drug moieties may impart their cytotoxic and cytostaticeffects by mechanisms including tubulin binding, DNA binding,proteasome, impairment of ribosome function, protein synthesis and/ortopoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibody.

Each antibody must be characterized separately, an appropriate linkerdesigned, and a suitable cytotoxic agent identified that retains itspotency upon delivery to tumor cells. One must consider the antigendensity on the cancer target and whether normal tissues express thetarget antigen. Other considerations include whether the entire ADC isinternalized upon binding the target; whether a cytostatic or cytotoxicdrug is preferable when considering possible normal tissue exposureand/or the type and stage of the cancer being treated; and, whether thelinker connecting the antibody to the drug payload is a cleavable or anon-cleavable linkage. Furthermore, the antibody to drug moietyconjugation ratio must be sufficient without compromising the bindingactivity of the antibody and/or the potency of the drug and withoutmodifying physicochemical properties of the ADC resulting on aggregationor deleterious properties regarding to the future development process ofthe compound.

An ADC is a complex biological molecule and the challenges to develop aneffective ADC remain a significant issue.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to anantibody-drug-conjugate of the following formula (I):Ab-(L-D)_(n)   (I)

or a pharmaceutically acceptable salt thereof,

wherein

Ab is a [Target] antibody or an antigen antibody, or a [Target] bindingfragment thereof or an antigen binding fragment thereof;

L is a linker;

D is a drug moiety of the following formula (II):

wherein:

R₁ is H or OH;

R₂ is a group: (C₁-C₆)alkyl, COOH, COO—((C₁-C₆)alkyl) or thiazolyl;

R₃ is H or a (C₁-C₆)alkyl group;

A is:

-   -   a group of formula -Het-Alk- wherein Alk is a (C₁-C₈)alkanediyl        group and is linked to NR₃, and Het is a heterocycle optionally        substituted by a (C₁-C₆)alkyl group and containing at least one        nitrogen atom, said nitrogen atom being linked to L, or    -   a group of formula -A_(a)-A_(b)- wherein A_(a) is linked to L        and is O or NR₉ with R₉ being H or (C₁-C₆)alkyl and A_(b) is        linked to NR₃ and is:        -   a (C₁-C₈)alkanediyl group,        -   a            —(CH₂CH₂X₁)_(a1)(CH₂CH₂X₂)_(a2)(CH₂CH₂X₃)_(a3)(CH₂CH₂X₄)_(a4)CH₂CH₂—            group with X₁, X₂, X₃ and X₄ each independently of one            another representing O or NR₈; a1, a2, a3 and a4 each            independently of one another representing 0 or 1; and R₈            representing H or a (C₁-C₆)alkyl group,        -   an aryl-(C₁-C₈)alkanediyl or heterocycle-(C₁-C₈)alkanediyl            group, said group being optionally substituted by a            (C₁-C₆)alkyl group, the aryl or heterocycle moiety being            linked to A_(a) and the (C₁-C₈)alkanediyl moiety being            linked to NR₃;

the wavy line indicates the point of attachment to L; and

n is 1 to 12.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein the [Target]or antigen of said [Target] antibody or antigen antibody according tothe invention, or a [Target] or an antigen binding fragment thereof, isselected from CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56, CD64,CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340,MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2, FAP, avintegrin, Mesothelin, EGFR, TAG-72, GD2, CAIX, 5T4, HER1, HER3, HER2,IGF-1R, Axl and their extra cellular membrane (ECD) fragment.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein the [Target]or antigen of said [Target] antibody or antigen antibody according tothe invention, or a [Target] or an antigen binding fragment thereof, isselected from the HER2, the IGF-1R and the protein Axl, preferably thehuman HER2, human IGF-1R and the human protein Axl, and their extracellular membrane (ECD) fragment.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein said Ab isan antibody, or an antigen binding fragment thereof, capable of bindingto the human IGF-1R selected from:

i) the antibodies 208F2, 212A11, 214F8, 219D6 and 213B10;

ii) the antibodies which compete for binding to IGF-1R with theantibodies of i); and

iii) the antibodies which bind to the same epitope of IGF-1R as theantibodies of i).

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein said Ab isan antibody, or an antigen binding fragment thereof, capable of bindingto the human IGF-1R selected from:

i) an antibody which comprises the three heavy chain CDRs of sequenceSEQ ID No. 1, 2 and 3 and the three light chain CDRs of sequence SEQ IDNo. 4, 5 and 6;

ii) an antibody which competes for binding to IGF-1R with the antibodyof i); and

iii) an antibody which binds to the same epitope of IGF-1R as theantibody of i).

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein said Ab isan antibody which comprises:

a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 whereinsaid sequence SEQ ID No. 33 comprises at least 1 back-mutation selectedfrom the residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76,77, 79, 82 or 95; and

b) a light chain variable domain (VL) of sequence SEQ ID No. 35, whereinsaid sequence SEQ ID No. 35 comprises at least 1 back-mutation selectedfrom the residues 22, 53, 55, 65, 71, 72, 77 or 87.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein said Ab isan antibody, or an antigen binding fragment thereof, capable of bindingto the human protein Axl selected from:

i) an antibody which comprises the three heavy chain CDRs of sequenceSEQ ID No. 59, 60 and 61 and the three light chain CDRs of sequence SEQID No. 56, 57 and 58;

ii) an antibody which competes for binding to Axl with the antibody ofi); and

iii) an antibody which binds to the same epitope of Axl as the antibodyof i).

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein said Ab isan antibody, or an antigen binding fragment thereof, capable of bindingto the human HER2, preferably consisting of Trastuzumab.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein L is alinker of the following formula (III):

wherein

L₂ is (C₄-C₁₀)cycloalkyl-carbonyl, (C₂-C₆)alkyl, (C₂-C₆)alkyl-carbonyl,

W is an amino acid unit; w is an integer comprised between 0 and 5;

Y is PAB-carbonyl with PAB being

y is 0 or 1;

the asterisk indicates the point of attachment to D; and

the wavy line indicates the point of attachment to Ab.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein L₂ is of thefollowing formula:

wherein

the asterisk indicates the point of attachment to (W)_(w); and

the wavy line indicates the point of attachment to the nitrogen atom ofthe maleimide moiety.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein w=0, or w=2and then (W)_(w) is selected from:

wherein

the asterisk indicates the point of attachment to (Y)_(y); and

the wavy line indicates the point of attachment to L₂.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein L isselected from:

wherein the asterisk indicates the point of attachment to D, and thewavy line indicates the point of attachment to Ab.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein A is a groupof formula -A_(a)-A_(b)- in which A_(a) is as defined in claim 1 andA_(b) is a group:

-   -   phenyl-(C₁-C₂)alkanediyl, or    -   heterocycle-(C₁-C₂)alkanediyl optionally substituted by a        (C₁-C₆)alkyl group (notably unsubstituted), the heterocycle        being a saturated, unsaturated or aromatic ring with 5 or 6        members comprising 1 or 2 nitrogen atoms, chosen in particular        from among pyridine, piperidine and imidazole, and being        preferably a pyridine.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein A is a groupof the following formula:

wherein:

R₉ is as defined in claim 1 and m is an integer comprised between 1 and8, and preferably wherein R₉=H or Me and m=1 or 2,

the wavy line indicates the point of attachment to L, and

the asterisk indicates the point of attachment to NR₃.

In an embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, wherein (L-D) isselected from:

wherein the wavy line indicates the point of attachment to Ab.

In another embodiment, the present invention relates to anantibody-drug-conjugate according to the invention, having the formulaselected from:

and the pharmaceutically acceptable salts thereof,

wherein Ab is a [Target] antibody or an antigen antibody, or a [Target]binding fragment thereof or an antigen binding fragment thereof.

In an embodiment, and according to the invention, said Ab is a [Target]antibody or an antigen antibody, or a [Target] binding fragment thereofor an antigen binding fragment thereof wherein said [Target] or antigenis selected from CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56, CD64,CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340,MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2, FAP, avintegrin, Mesothelin, EGFR, TAG-72, GD2, CAIX, 5T4, HER1, HER3, HER2,IGF-1R,Axl and their extra cellular membrane (ECD) fragment.

In an embodiment, and according to the invention, said Ab is a [Target]antibody or antigen antibody, or a [Target] or an antigen bindingfragment thereof, wherein said [Target] or antigen is selected from theHER2, the IGF-1R and the protein Axl, preferably the human HER2, thehuman IGF-1R and the human protein Axl, and their extra cellularmembrane (ECD) fragment.

In an embodiment, and according to the invention, said Ab is anantibody, or an antigen binding fragment thereof, and is selected from:

a) an Ab, or an antigen binding fragment thereof, capable of binding tothe human IGF-1R selected from:

-   -   i) the antibodies 208F2, 212A11, 214F8, 219D6 and 213B10;    -   ii) the antibodies which compete for binding to IGF-1R with the        antibodies of i); and    -   iii) the antibodies which bind to the same epitope of IGF-1R as        the antibodies of i),        b) an Ab, or an antigen binding fragment thereof, capable of        binding to the human IGF-1R selected from:    -   i) an antibody which comprises the three heavy chain CDRs of        sequence SEQ ID No. 1, 2 and 3 and the three light chain CDRs of        sequence SEQ ID No. 4, 5 and 6;    -   ii) an antibody which competes for binding to IGF-1R with the        antibody of i); and    -   iii) an antibody which binds to the same epitope of IGF-1R as        the antibody of i),        c) an Ab which comprises:    -   i) a heavy chain variable domain (VH) of sequence SEQ ID No. 33        wherein said sequence SEQ ID No. 33 comprises at least 1        back-mutation selected from the residues 20, 34, 35, 38, 48, 50,        59, 61, 62, 70, 72, 74, 76, 77, 79, 82 or 95; and    -   ii) a light chain variable domain (VL) of sequence SEQ ID No.        35, wherein said sequence SEQ ID No. 35 comprises at least 1        back-mutation selected from the residues 22, 53, 55, 65, 71, 72,        77 or 87,        d) an Ab, or an antigen binding fragment thereof, capable of        binding to the human protein Axl selected from:    -   i) an antibody which comprises the three heavy chain CDRs of        sequence SEQ ID No. 59, 60 and 61 and the three light chain CDRs        of sequence SEQ ID No. 56, 57 and 58;    -   ii) an antibody which competes for binding to Axl with the        antibody of i); and    -   iii) an antibody which binds to the same epitope of Axl as the        antibody of i), and        e) an Ab, or an antigen binding fragment thereof, capable of        binding to the human HER2, preferably consisting of Trastuzumab        or Pertuzumab.

In an embodiment, and according to the invention, n is 2 or n is 4.

In another embodiment, the present invention is directed to anantibody-drug-conjugate according to the present invention for use as amedicament.

In another embodiment, the present is directed to a compositioncomprising at least one antibody-drug-conjugate according to the presentinvention.

In an embodiment, the composition according to the present inventionfurther comprises a pharmaceutically acceptable vehicle.

In another embodiment, the present invention is directed to acomposition according to the present invention for use in the treatmentof a [Target]- or an antigen-expressing cancer, said Target]- or anantigen being preferably selected from CD19, CD20, CD22, CD25, CD30,CD33, CD40, CD56, CD64, CD70, CD74, CD79, CD105, CD138, CD174, CD205,CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2,EphA2, FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2, CAIX, 5T4, HER1,HER3, HER2, IGF-1R,Axl and their extra cellular membrane (ECD) fragment,more preferably from the HER2, the IGF-1R and the protein Axl, also morepreferably the human HER2, the human IGF-1R and the human protein Axl,and their extra cellular membrane (ECD) fragment.

In an embodiment, said [Target]- or antigen-expressing cancer is acancer chosen from breast, colon, esophageal carcinoma, hepatocellular,gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate,rhabdomyosarcoma, renal, thyroid, uterine endometrial cancer,mesothelioma, oral squamous carcinoma, Kaposi sarcoma, acute leukemia,colorectal carcinoma, melanoma, pancreatic ductal adenocarcinoma and anydrug resistant cancer.

In an embodiment, the present invention relates to a method for thetreatment of a [Target]- or antigen-expressing cancer in a subject inneed thereof, comprising administering to the subject an effectiveamount of at least one antibody-drug-conjugate according to the presentinvention or of a composition according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION I—The Antibody (Ab)

The terms “antibody”, “antibodies” “ab”, “MAb” or “immunoglobulin” areused interchangeably in the broadest sense and include monoclonalantibodies, preferably isolated, engineered or recombinant antibodies(e.g., full length or intact monoclonal antibodies), polyclonalantibodies, multivalent antibodies or multispecific antibodies (e.g.,bispecific antibodies) and also antibody fragment thereof, so long asthey exhibit the desired biological activity.

In an embodiment, the antibody of the invention consists of arecombinant antibody. The term “recombinant antibody” refers to anantibody that results from the expression of recombinant DNA withinliving cells. A recombinant antibody of the invention is obtained byusing laboratory methods of genetic recombination, well known by aperson skilled in the art, creating DNA sequences that would not befound in biological organisms.

In another embodiment, the antibody of the invention consists of achemically synthesized antibody.

More particularly, such a molecule consists of a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain comprises a heavy chain variableregion (or domain) (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, CH1, CH2 and CH3. Each light chain comprises a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region comprises one domain,CL. The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g. effector cells) and the first component(Clq) of the classical complement system.

By “antigen binding fragment” or “target binding fragment” of anantibody according to the invention, it is intended to indicate anypeptide, polypeptide, or protein retaining the ability to bind to thetarget (also generally referred as antigen) of the antibody. andcomprising an amino acid sequence of at least 10 contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least contiguous 80 aminoacid residues, at least contiguous 90 amino acid residues, at leastcontiguous 100 amino acid residues, at least contiguous 125 amino acidresidues, at least 150 contiguous amino acid residues, at leastcontiguous 175 amino acid residues, at least contiguous 200 amino acidresidues, or at least contiguous 250 amino acid residues of the aminoacid sequence of the antibody.

In an embodiment, such “antigen binding fragments” are selected in thegroup consisting of Fv, scFv (sc for single chain), Fab, F(ab′)₂, Fab′,scFv-Fc fragments or diabodies, or any fragment of which the half-lifetime would have been increased by chemical modification, such as theaddition of poly(alkylene) glycol such as poly(ethylene) glycol(“PEGylation”) (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG,F(ab′)₂-PEG or Fab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or byincorporation in a liposome, said fragments having at least one of thecharacteristic CDRs of the antibody according to the invention.Preferably, said “antigen binding fragments” will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthe affinity of the antibody from which it is descended, with respect tothe target.

By “binding”, “binds”, or the like, it is intended that antibody, or anyantigen binding fragment thereof, forms a complex with an antigen thatis relatively stable under physiologic conditions. Specific binding canbe characterized by an equilibrium dissociation constant of at leastabout 1·10⁻⁶ M. Methods for determining whether two molecules bind arewell known in the art and include, for example, equilibrium dialysis,surface plasmon resonance, radiolabelled assays and the like. For theavoidance of doubt, it does not mean that the said antibody could notbind or interfere, at a low level, to another antigen. Nevertheless, asa preferred embodiment, the said antibody binds only to the saidantigen.

As used in the present specification, the expression “[Target] antibody”should be interpreted as similar to “anti-[Target] antibody” and meansan antibody capable of binding to the [Target].

The expression “Target” or [Target] shall be interpreted as any moleculepresent at the surface of cells, preferably tumoral cells, morepreferably mammals and human cells, and which can be used for drugdelivery. Preferably, the Target is specifically express or overexpresson the surface of tumoral cells in comparison with normal cells.

More particularly, the Target can be selected from CD19, CD20, CD22,CD25, CD30, CD33, CD40, CD56, CD64, CD70, CD74, CD79, CD105, CD138,CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B,TMEFF2, EphB2, EphA2, FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2,CAIX, 5T4, HER1, HER3, HER2, IGF-1R or Axl (B. A. Teicher, CurrentCancer Drug Targets, 2009, 9, 982-1004).

As preferred Target, it can be mentioned HER2, IGF-1R and Axl,preferably human HER2, IGF-1R and Axl.

As non limitative example, the expression “HER2 antibody”, “IGF-1Rantibody” or “Axl antibody” should be interpreted as similar to“anti-HER2 antibody”, “anti-IGF-1R antibody” or “anti-Axl antibody” andmeans an antibody capable of binding to HER2, IGF-1R or Axl,respectively.

In the present application, the epitope of the antibody ispreferentially localized into the extracellular domain of the Target.

In a particular embodiment, the antibody, or any antigen bindingfragment thereof, is capable of binding to the Target with an EC₅₀comprised between 10·10⁻¹⁰ to 1×10⁻¹⁰, and more preferentially between8×10⁻¹⁰ to 2×10⁻¹⁰

The term half maximal effective concentration (EC₅₀) corresponds to theconcentration of a drug, antibody or toxicant which induces a responsehalfway between the baseline and maximum after some specified exposuretime. It is commonly used as a measure of drug's potency. The EC₅₀ of agraded dose response curve therefore represents the concentration of acompound where 50% of its maximal effect is observed. The EC₅₀ of aquantal dose response curve represents the concentration of a compoundwhere 50% of the population exhibits a response, after specifiedexposure duration. Concentration measures typically follow a sigmoidalcurve, increasing rapidly over a relatively small change inconcentration. This can be determined mathematically by derivation ofthe best-fit line.

As a preferred embodiment, the EC₅₀, determined in the presentinvention, characterizes the potency of antibody to bind on the TargetECD exposed on human tumor cells. The EC₅₀ parameter is determined usingFACS analysis. The EC₅₀ parameter reflects the antibody concentrationfor which 50% of the maximal binding on the human Target expressed ontumor cells is obtained. Each EC₅₀ value was calculated as the midpointof the dose response curve using a four-parameter regression curvefitting program (Prism Software). This parameter has been selected as tobe representative of physiological/pathological conditions.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The competition for binding to the Target can be determined by anymethods or techniques known by the person skilled in the art such as,without limitation, radioactivity, Biacore, ELISA, Flow cytometry, etc.As “which competes for binding to the Target” it is meant a competitionof at least 20%, preferentially at least 50% and more preferentially atleast 70%.

The determination of the binding to the same epitope can be determinedby any methods or techniques known by the person skilled in the art suchas, without limitation in the examples, radioactivity, Biacore, ELISA,Flow cytometry. As “which bind to the same epitope of the Target”, it ismeant a competition of at least 20%, preferentially at least 50% andmore preferentially at least 70%.

As above mentioned, and contrary to the general knowledge, the presentinvention focuses on specific antibodies presenting a high ability to beinternalized following their binding to the Target. As used herein, anantibody that “is internalized” or that “internalized” (the twoexpressions being similar) is one that is taken up by (meaning it“enters”) the cell upon binding to the Target on a mammalian cell. Suchan antibody is interesting as part of the ADC, so it addresses thelinked cytotoxic into the targeted cancer cells. Once internalized thecytotoxic triggers cancer cell death.

Important keys to success with ADC therapy are thought to be the targetantigen specificity and the internalization of the antigen-antibodycomplexes into the cancer cells. Obviously non-internalizing antigensare less effective than internalizing antigens to delivers cytotoxicagents. Internalization processes are variable across antigens anddepend on multiple parameters that can be influenced by antibodies.

In the ADC, the cytotoxic confers the cytotoxic activity and the usedantibody is responsible for the specificity against cancer cells, aswell as a vector for entering within the cells to correctly address thecytotoxic. Thus to improve the ADC, the antibody must exhibit highability to internalize into the targeted cancer cells. The efficiency ofthe antibody mediated internalisation differs significantly depending onthe epitope targeted. Selection of potent internalizing antibodiesrequires various experimental data studying not only the Targetdownregulation but also following the antibody penetration into thecells.

In a preferred embodiment, the internalization of the antibody accordingto the invention can be evaluated by immunofluorescence or FACS (FlowCytometry) (as exemplified hereinafter in the present application) orany method or process known by the person skilled in the art specificfor the internalization mechanism. In a preferred embodiment, theantibody according to the invention can induce internalization afterbinding to the Target of at least 30%, preferentially 50% and morepreferentially 80%.

The complex Target/antibody is internalized after binding of theantibody to the ECD of said Target, and a reduction in the quantity ofTarget at the surface of the cells is induced. This reduction can bequantified by any method known by the person skilled in the art(western-blot, FACS, immunofluorescence, etc).

In one embodiment, this reduction, thus reflecting the internalization,can be preferably measured by FACS and expressed as the difference ordelta between the Mean Fluorescence Intensity (MFI) measured at 4° C.with the MFI measured at 37° C. after 4 hours incubation with theantibody.

As non limitative example, this delta is determined based on MFIsobtained with untreated cells and cells treated with the antibody usingi) cancer cells, such as for example MCF,7 after a 4 hour incubationperiod with the antibody herein described and ii) a secondary antibodylabelled with Alexa488. This parameter is defined as calculated with thefollowing formula: Δ(MFI_(4° C.)-MFI_(37° C.)). Other embodiments willbe detailed in the following examples.

This difference between MFIs reflects the Target downregulation as MFIsare proportional to Target expressed on the cell-surface.

In an advantageous aspect, the antibodies consist of antibodiestriggering, as an example, a Δ(MFI_(4° C.)-MFI_(37° C.)) on cancer cell,such as MCF-7, of at least 280, preferably of at least 400.

In more details, the above mentioned delta can be measured according tothe following process, which must be considered as an illustrative andnon limitative example:

-   -   a) Treating and incubating tumor cells of interest with the        antibody of the invention in either cold (4° C.) or warm (37°        C.) complete culture medium;    -   b) Treating the treated cells of step a) and, in parallel,        untreated cells with a secondary antibody;    -   c) Measuring the MFI (representative of the quantity of IGF-1R        present at the surface) for the treated and the non treated        cells with a secondary labeled antibody capable of binding to        the antibody of the invention; and    -   d) Calculating the delta as the subtraction of the MFI obtained        with the treated cells from the MFI obtained with the non        treated cells.

From this delta MFI, an internalization percentage can be determined as:100×(MFI_(4° C.)-MFI_(37° C.))/MFI_(4° C.).

The antibodies, according to the invention, present, preferably, aninternalization percentage comprised between 50% and 99%, 70% and 90%,preferentially between 75% and 87%.

A particular advantage of the antibodies herein described relies ontheir rate of internalization.

It is generally known that, for an ADC, it is desirable that the usedantibodies exhibit a rapid rate of internalization, preferably within 24hours from administration of the antibody and, more preferably within 12hours and, even more preferably within 6 hours.

In the present invention, the internalization rate, also referred ascell surface bound antibody decrease or cell surface antibody decay, isexpressed as t1/2 (half life) and corresponds as the time necessary toobtain a decrease of 50% of the ΔMFI (this aspect will be clearlyunderstood regarding the following examples).

A particular advantage is that the antibodies of the invention have at1/2 comprised between 5 and 25 minutes, and preferentially between 10and 20 minutes.

In the following specification, two preferred Target will beexemplified.

For these two preferred Target, by “antibody according to the invention”or “antibody of the invention” it must be understood: “antibody of theADC according to the invention” or “antibody of the ADC of theinvention” respectively.

I.1: IGF-1R Antibodies

A particular embodiment of the invention relates to an ADC wherein theantibody Ab comprises three heavy chain CDRs with CDR-H2 of sequence SEQID No. 2 and CDR-H3 of sequence SEQ ID No. 3, and three light chain CDRswith CDR-L2 of sequence SEQ ID No. 5.

A particular embodiment of the invention relates to an ADC wherein theantibody Ab comprises the three heavy chain CDRs of sequences SEQ IDNos. 1, 2 and 3 and the three light chain CDRs of sequences SEQ ID Nos.4, 5 and 6.

An embodiment of the ADC comprises an antibody comprising the threeheavy chain CDRs comprising the sequences SEQ ID Nos. 1, 2 and 3, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID Nos. 1, 2 and 3; and the three light chain CDRscomprising the sequences SEQ ID Nos. 4, 5 and 6, or any sequenceexhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity withSEQ ID Nos. 4, 5 and 6.

In another embodiment, the antibody, or any antigen binding fragmentthereof, comprises the three light chain CDRs comprising the sequencesSEQ ID Nos. 1, 2 and 3; and the three heavy chain CDRs comprising thesequences SEQ ID Nos. 4, 5 and 6.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cystein 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

It must be understood that, without contradictory specification in thepresent specification, complementarity-determining regions or CDRs, meanthe hypervariable regions of the heavy and light chains ofimmunoglobulins as defined according to the IMGT numbering system.

Nevertheless, CDRs can also be defined according to the Kabat numberingsystem (Kabat et al., Sequences of proteins of immunological interest,5^(th) Ed., U.S. Department of Health and Human Services, NIH, 1991, andlater editions). There are three heavy chain CDRs and three light chainCDRs. Here, the terms “CDR” and “CDRs” are used to indicate, dependingon the case, one or more, or even all, of the regions containing themajority of the amino acid residues responsible for the antibody'sbinding affinity for the antigen or epitope it recognizes. In order tosimplify the reading of the present application, the CDRs according toKabat are not defined. Nevertheless, it would be obvious for the personskilled in the art, using the definition of the CDRs according to IMGT,to define the CDRs according to Kabat.

In the sense of the present invention, the “identity” or “percentageidentity” between two sequences of nucleic acids or amino acids meansthe percentage of identical nucleotides or amino acid residues betweenthe two sequences to be compared, obtained after optimal alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly along their length. The comparisonof two nucleic acid or amino acid sequences is traditionally carried outby comparing the sequences after having optimally aligned them, saidcomparison being able to be conducted by segment or by using an“alignment window”. Optimal alignment of the sequences for comparisoncan be carried out, in addition to comparison by hand, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48:443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or by the comparison softwareBLAST NR or BLAST P).

Percentage identity is calculated by determining the number of positionsat which the amino acid nucleotide or residue is identical between thetwo sequences, preferably between the two complete sequences, dividingthe number of identical positions by the total number of positions inthe alignment window and multiplying the result by 100 to obtain thepercentage identity between the two sequences.

For example, the BLAST program, “BLAST 2 sequences” (Tatusova et al.,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol., 1999, Lett. 174:247-250) available on thesite http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with thedefault parameters (notably for the parameters “open gap penalty”: 5,and “extension gap penalty”: 2; the selected matrix being for examplethe “BLOSUM 62” matrix proposed by the program); the percentage identitybetween the two sequences to compare is calculated directly by theprogram.

For the amino acid sequence exhibiting at least 80%, preferably 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated.

As a non-limiting example, table 1 below summarizes the possiblesubstitutions likely to be carried out without resulting in asignificant modification of the biological activity of the correspondingmodified antibody; inverse substitutions are naturally possible underthe same conditions.

TABLE 1 Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly(G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met(M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) TyrTyr (Y) Phe, Trp Val (V) Leu, Ala

A particular aspect of the invention is that the antibody, does not bindto the Insulin receptor (IR). This aspect is of interest as the antibodyherein described will not have any negative impact on the IR, meaningthe Insulin metabolism.

Still another advantageous aspect of the antibody of the ADC of theinvention is that it is capable of binding not only to the human IGF-1Rbut also to the monkey IGF-1R, and more particularly to the cynomolgusIGF-1R. This aspect is also of interest as it will facilitate thetoxicity assessment required for clinical trials.

In still another embodiment, the antibody of the ADC of the inventionconsists of a monoclonal antibody.

The term “monoclonal antibody” or “Mab” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e. the individual antibodies of the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single epitope. Such monoclonal antibody may beproduced by a single clone of B cells or hybridoma. Monoclonalantibodies may also be recombinant, i.e. produced by protein engineeringor chemical synthesis. Monoclonal antibodies may also be isolated fromphage antibody libraries. In addition, in contrast with preparations ofpolyclonal antibodies which typically include various antibodiesdirected against various determinants, or epitopes, each monoclonalantibody is directed against a single epitope of the antigen.

The monoclonal antibody herein includes murine, chimeric and humanizedantibody, such as described after.

The antibody is preferably derived from an hybridoma of murine originfiled within the French collection for microorganism cultures (CNCM,Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15,France), said hybridoma being obtained by the fusion of Balb/C immunizedmice splenocytes/lymphocytes and cells of the myeloma Sp 2/O-Ag 14 cellline.

In an embodiment, the IGF-1R antibody of the ADC of the inventionconsists of a murine antibody, then referred as m[name of the antibody].

In an embodiment, the IGF-1R antibody consists of a chimeric antibody,then referred as c[name of the antibody].

In an embodiment, the IGF-1R antibody consists of a humanized antibody,then referred as hz[name of the antibody].

For the avoidance of doubt, in the following specification, theexpressions “IGF-1R antibody” and “[name of the antibody]” are similarand include (without contrary specification) the murine, the chimericand the humanized versions of the said IGF-1R antibody or of the said“[name of the antibody]”. When necessary, the prefix m-(murine), c-(chimeric) or hz- (humanized) is used.

For more clarity, the following table 2 illustrates the CDR sequences,defined according to IMGT, for the preferred antibodies.

TABLE 2 Heavy chain Light chain SEQ ID No. Consensus CDR-H1 1 CDR-H2 2CDR-H3 3 CDR-L1 4 CDR-L2 5 CDR-L3 6 208F2 CDR-H1 7 CDR-H2 2 CDR-H3 3CDR-L1 9 CDR-L2 5 CDR-L3 11 212A11 CDR-H1 7 CDR-H2 2 CDR-H3 3 CDR-L1 10CDR-L2 5 CDR-L3 11 214F8 CDR-H1 7 & CDR-H2 2 213B10 CDR-H3 3 CDR-L1 9CDR-L2 5 CDR-L3 12 219D6 CDR-H1 8 CDR-H2 2 CDR-H3 3 CDR-L1 9 CDR-L2 5CDR-L3 11

It will be obvious for the man skilled in the art that any combinationof 6 CDRs as above described should be considered as part of the presentinvention.

As can be observed from this table 2, all the antibodies hereindescribed have the same sequences for the CDR-H2, CDR-H3 and CDR-L2,this property being of particular interest as above described.

A specific aspect relates to a murine antibody characterized in thatsaid antibody also comprises light chain and heavy chain constantregions derived from an antibody of a species heterologous with themouse, notably man.

Another specific aspect relates to a chimeric (c) antibody characterizedin that said antibody also comprises light chain and heavy chainconstant regions derived from an antibody of a species heterologous withthe mouse, notably human.

A chimeric antibody is one containing a natural variable region (lightchain and heavy chain) derived from an antibody of a given species incombination with constant regions of the light chain and the heavy chainof an antibody of a species heterologous to said given species.

The chimeric antibodies can be prepared by using the techniques ofrecombinant genetics. For example, the chimeric antibody could beproduced by cloning recombinant DNA containing a promoter and a sequencecoding for the variable region of a nonhuman monoclonal antibody of theADC of the invention, notably murine, and a sequence coding forheterologous species antibody constant region, preferably human. Achimeric antibody the ADC according to the invention coded by one suchrecombinant gene could be, for example, a mouse-human chimera, thespecificity of this antibody being determined by the variable regionderived from the murine DNA and its isotype determined by the constantregion derived from human DNA.

In a preferred, but not limitative, embodiment, the antibody of the ADCof the invention is selected from:

a) an antibody comprising a heavy chain variable domain of sequence SEQID No. 13 or any sequence exhibiting at least 80% identity with SEQ IDNo. 13 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and11;

b) an antibody comprising a heavy chain variable domain of sequence SEQID No. 14 or any sequence exhibiting at least 80% identity with SEQ IDNo. 14 and the three light chain CDRs of sequences SEQ ID Nos. 10, 5 and11;

c) an antibody comprising a heavy chain variable domain of sequence SEQID No. 15 or any sequence exhibiting at least 80% identity with SEQ IDNo. 15 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and12;

d) an antibody comprising a heavy chain variable domain of sequence SEQID No. 16 or any sequence exhibiting at least 80% identity with SEQ IDNo. 16 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and11; and

e) an antibody comprising a heavy chain variable domain of sequence SEQID No. 17 or any sequence exhibiting at least 80% identity with SEQ IDNo. 17 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and12.

By “any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and98% identity with SEQ ID No. 13 to 17”, its is intended to designate thesequences exhibiting the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 13 to 17 outside thesequences corresponding to the CDRs (i.e. SEQ ID No. 1, 2 and 3).

In another preferred, but not limitative, embodiment, the antibody ofthe ADC of the invention is selected from:

a) an antibody comprising a light chain variable domain of sequence SEQID No. 18 or any sequence exhibiting at least 80% identity with SEQ IDNo. 18 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3;

b) an antibody comprising a light chain variable domain of sequence SEQID No. 19 or any sequence exhibiting at least 80% identity with SEQ IDNo. 19 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3;

c) an antibody comprising a light chain variable domain of sequence SEQID No. 20 or any sequence exhibiting at least 80% identity with SEQ IDNo. 20 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3;

d) an antibody comprising a light chain variable domain of sequence SEQID No. 21 or any sequence exhibiting at least 80% identity with SEQ IDNo. 21 and the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and3; and

e) an antibody comprising a light chain variable domain of sequence SEQID No. 22 or any sequence exhibiting at least 80% identity with SEQ IDNo. 22 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3.

By “any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and98% identity with SEQ ID No. 18 to 22”, its is intended to designate thesequences exhibiting the three light chain CDRs SEQ ID Nos. 4, 5 and 6and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 18 to 22 outside thesequences corresponding to the CDRs (i.e. SEQ ID No. 4, 5 and 6).

An embodiment of the invention relates to an ADC wherein Ab is anantibody selected from:

a) an antibody comprising a heavy chain variable domain of sequence SEQID No. 13 or any sequence exhibiting at least 80% identity with SEQ IDNo. 13 and a light chain variable domain of sequence SEQ ID No. 18 orany sequence exhibiting at least 80% identity with SEQ ID No. 18;

b) an antibody comprising a heavy chain variable domain of sequence SEQID No. 14 or any sequence exhibiting at least 80% identity with SEQ IDNo. 14 and a light chain variable domain of sequence SEQ ID No. 19 orany sequence exhibiting at least 80% identity with SEQ ID NO. 19;

c) an antibody comprising a heavy chain variable domain of sequence SEQID No. 15 or any sequence exhibiting at least 80% identity with SEQ IDNo. 15 and a light chain variable domain of sequence SEQ ID No. 20 orany sequence exhibiting at least 80% identity with SEQ ID No. 20;

d) an antibody comprising a heavy chain variable domain of sequence SEQID No. 16 or any sequence exhibiting at least 80% identity with SEQ IDNo. 16 and a light chain variable domain of sequence SEQ ID No. 21 orany sequence exhibiting at least 80% identity with SEQ ID No. 21; and

e) an antibody comprising a heavy chain variable domain of sequence SEQID No. 17 or any sequence exhibiting at least 80% identity with SEQ IDNo. 17 and a light chain variable domain of sequence SEQ ID No. 22 orany sequence exhibiting at least 80% identity with SEQ ID No. 22.

Chimeric antibodies herein described can be also characterized by theconstant domain and, more particularly, said chimeric antibodies can beselected or designed such as, without limitation, IgG1, IgG2, IgG3, IgM,IgA, IgD or IgE. More preferably, in the context of the presentinvention, said chimeric antibodies are IgG1 or IgG4.

An embodiment of the invention relates to an ADC wherein Ab is achimeric antibody comprising variable domains VH and VL as abovedescribed in the format IgG1. More preferably, said chimeric antibodycomprises a constant domain for the VH of sequence SEQ ID No. 43 and aKappa domain for the VL of sequence SEQ ID No. 45.

An embodiment of the invention relates to an ADC wherein Ab is achimeric antibody comprising variable domains VH and VL as abovedescribed in the format IgG4. More preferably, said chimeric antibodycomprises a constant domain for the VH of sequence SEQ ID No. 44 and aKappa domain for the VL of sequence SEQ ID No. 45.

In another preferred, but not limitative, embodiment, the antibody ofthe ADC of the invention is selected from:

a) an antibody comprising or consisting of a heavy chain of sequence SEQID No. 23 or any sequence exhibiting at least 80% identity with SEQ IDNo. 23 and a light chain of sequence SEQ ID No. 28 or any sequenceexhibiting at least 80% identity with SEQ ID No. 28;

b) an antibody comprising or consisting of a heavy chain of sequence SEQID No. 24 or any sequence exhibiting at least 80% identity with SEQ IDNo. 24 and a light chain of sequence SEQ ID No. 29 or any sequenceexhibiting at least 80% identity with SEQ ID No. 29;

c) an antibody comprising or consisting of a heavy chain of sequence SEQID No. 25 or any sequence exhibiting at least 80% identity with SEQ IDNo. 25 and a light chain of sequence SEQ ID No. 30 or any sequenceexhibiting at least 80% identity with SEQ ID No. 30;

d) an antibody comprising or consisting of a heavy chain of sequence SEQID No. 26 or any sequence exhibiting at least 80% identity with SEQ IDNo. 26 and a light chain of sequence SEQ ID No. 31 or any sequenceexhibiting at least 80% identity with SEQ ID No. 31; and

e) an antibody comprising or consisting of a heavy chain of sequence SEQID No. 27 or any sequence exhibiting at least 80% identity with SEQ IDNo. 27 and a light chain of sequence SEQ ID No. 32 or any sequenceexhibiting at least 80% identity with SEQ ID No. 32.

For more clarity, the following table 3 illustrates the sequences of theVH and VL, respectively, for the preferred chimeric antibodies.

TABLE 3 SEQ Heavy Chain Light chain ID No. c208F2 Variable domain (VH)13 Variable domain (VL) 18 Full length 23 Full length 28 c212A11Variable domain (VH) 14 Variable domain (VL) 19 Full length 24 Fulllength 29 c214F8 Variable domain (VH) 15 Variable domain (VL) 20 Fulllength 25 Full length 30 c219D6 Variable domain (VH) 16 Variable domain(VL) 21 Full length 26 Full length 31 c213B10 Variable domain (VH) 17Variable domain (VL) 22 Full length 27 Full length 32

Yet another specific aspect of the present invention relates to an ADCwherein Ab is a humanized antibody characterized in that the constantregions of the light chain and the heavy chain derived from humanantibody are, respectively, the lambda or kappa region and the gamma-1,gamma-2 or gamma-4 region.

“Humanized antibodies” means an antibody that contains CDR regionsderived from an antibody of nonhuman origin, the other parts of theantibody molecule being derived from one (or several) human antibodies.In addition, some of the skeleton segment residues (called FR) can bemodified to preserve binding affinity.

The humanized antibodies or fragments of same can be prepared bytechniques known to a person skilled in the art. Such humanizedantibodies are preferred for their use in methods involving in vitrodiagnoses or preventive and/or therapeutic treatment in vivo. Otherhumanization techniques, also known to a person skilled in the art, suchas, for example, the “CDR grafting” technique described by PDL inpatents EP 0 451 216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or U.S.Pat. No. 5,530,101, U.S. Pat. No. 6,180,370, U.S. Pat. No. 5,585,089 andU.S. Pat. No. 5,693,761. U.S. Pat. Nos. 5,639,641 or 6,054,297,5,886,152 and 5,877,293 can also be cited.

As a particular embodiment of the invention, and as it will beexplicated in more details in the examples after, it is herein describedan antibody consisting of the hz208F2. Such humanization can also beapplied to the other antibodies part of the present invention.

In a preferred embodiment, the antibody of the ADC according to thepresent invention comprises a heavy chain variable domain (VH) having:

i) the CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7, 2 and 3,respectively, and

ii) the FR1, FR2 and FR3 derived from the human germline IGHV1-46*01(SEQ ID No. 46), and

iii) the FR4 derived from the human germline IGHJ4*01 (SEQ ID No. 48).

In a preferred embodiment, the antibody of the ADC according to thepresent invention comprises a light chain variable domain (VL) having:

i) the CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5 and 11,respectively, and

ii) the FR1, FR2 and FR3 derived from the human germline IGKV1-39*01(SEQ ID No. 47), and

iii) the FR4 derived from the human germline IGKJ4*01 (SEQ ID No. 49).

In a preferred, but not limitative, embodiment of the invention, theantibody of the ADC comprises:

a) a heavy chain having CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ IDNos. 7, 2 and 3, respectively, and FR1, FR2 and FR3 derived from thehuman germline IGHV1-46*01 (SEQ ID No. 46), and the FR4 derived from thehuman germline IGHJ4*01 (SEQ ID No. 48); and

b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ IDNos. 9, 5 and 11, respectively, and FR1, FR2 and FR3 derived from thehuman germline IGKV1-39*01 (SEQ ID No. 47), and the FR4 derived from thehuman germline IGKJ4*01 (SEQ ID No. 49).

In an embodiment, the antibody of the ADC according to the inventioncomprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33and a light chain variable domain (VL) of sequence SEQ ID No. 35. Saidhumanized antibody will be called thereinafter hz208F2 (“Variant 1” or“Var. 1”).

In another embodiment, the antibody of the ADC according to the presentinvention comprises a heavy chain variable domain (VH) of sequence SEQID No. 33 wherein said sequence SEQ ID No. 33 comprises at least 1back-mutation selected from the residues 20, 34, 35, 38, 48, 50, 59, 61,62, 70, 72, 74, 76, 77, 79, 82 or 95.

By the expressions “back-mutation” or “back mutation” it is meant amutation or replacement of the human residue present in the germline bythe corresponding residue initially present in the murine sequence.

In another embodiment, the antibody of the ADC according to the presentinvention comprises a heavy chain variable domain (VH) of sequence SEQID No. 33 wherein said sequence SEQ ID No. 33 comprises 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 back-mutations selected fromthe residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79,82 or 95.

For more clarity, the following table 4 illustrates the preferredback-mutations.

TABLE 4 N^(o) résidu 20 34 35 38 48 50 59 61 Murin M I Y K L W K Nhumain V M H R M I S A N^(o) résidu 62 70 72 74 76 77 79 82 95 Murin E LA K S N A F F humain Q M R T T S V E YIn an embodiment, the antibody of the ADC according to the presentinvention comprises a light chain variable domain (VL) of sequence SEQID No. 35, wherein said sequence SEQ ID No. 35 comprises at least 1back-mutation selected from the residues 22, 53, 55, 65, 71, 72, 77 or87.

In an embodiment, the antibody of the ADC according to the presentinvention comprises a light chain variable domain (VL) of sequence SEQID No. 35, wherein said sequence SEQ ID No. 35 comprises 2, 3, 4, 5, 6,7 or 8 back-mutations selected from the residues 22, 53, 55, 65, 71, 72,77 or 87.

In another embodiment, the antibody of the ADC according to the presentinvention comprises:

a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 whereinsaid sequence SEQ ID No. 33 comprises at least 1 back-mutation selectedfrom the residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76,77, 79, 82 or 95; and

b) a light chain variable domain (VL) of sequence SEQ ID No. 35, whereinsaid sequence SEQ ID No. 35 comprises at least 1 back-mutation selectedfrom the residues 22, 53, 55, 65, 71, 72, 77 or 87.

For more clarity, the following table 5 illustrates the preferredback-mutations.

TABLE 5 N^(o) résidu 22 53 55 65 71 72 77 87 Murin S R H R Y S N Fhumain T S Q S F T S Y

In such an embodiment, the antibody of the ADC according to theinvention comprises all the back-mutations above mentioned andcorresponds to an antibody comprising a heavy chain variable domain (VH)of sequence SEQ ID No. 34 and a light chain variable domain (VL) ofsequence SEQ ID No. 36. Said humanized antibody will be calledthereinafter hz208F2 (“Variant 3” or “Var. 3”).

In another embodiment, all the humanized forms comprised between theVariant 1 and the Variant 3 are also encompassed by the presentinvention. In other words, the antibody of the ADC according to theinvention corresponds to an antibody comprising a heavy chain variabledomain (VH) of “consensus” sequence SEQ ID No. 41 and a light chainvariable domain (VL) of “consensus” sequence SEQ ID No. 42. Saidhumanized antibody, as a whole, will be called thereinafter hz208F2(“Variant2” or “Var.2”).

In a preferred, but not limitative, embodiment, the antibody of the ADCof the invention is selected from:

a) an antibody comprising a heavy chain variable domain of sequence SEQID No. 33 or any sequence exhibiting at least 80% identity with SEQ IDNo. 33 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and11; or

b) an antibody comprising a heavy chain variable domain of sequence SEQID No. 34 or any sequence exhibiting at least 80% identity with SEQ IDNo. 34 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and11.

By “any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and98% identity with SEQ ID No. 33 or 34”, its is intended to designate thesequences exhibiting the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 33 or 34 outside thesequences corresponding to the CDRs (i.e. SEQ ID Nos. 1, 2 and 3).

In a preferred, but not limitative, embodiment, the antibody of the ADCof the invention is selected from:

a) an antibody comprising a light chain variable domain of sequence SEQID No. 35 or any sequence exhibiting at least 80% identity with SEQ IDNo. 35 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3; and

b) an antibody comprising a heavy chain variable domain of sequence SEQID No. 36 or any sequence exhibiting at least 80% identity with SEQ IDNo. 36 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and3.

By “any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and98% identity with SEQ ID No. 35 or 36”, its is intended to designate thesequences exhibiting the three light chain CDRs SEQ ID Nos. 4, 5 and 6and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 35 or 36 outside thesequences corresponding to the CDRs (i.e. SEQ ID Nos. 4, 5 and 6).

Humanized antibodies herein described can be also characterized by theconstant domain and, more particularly, said humanized antibodies can beselected or designed such as, without limitation, IgG1, IgG2, IgG3, IgM,IgA, IgD or IgE. More preferably, in the context of the presentinvention, said humanized antibodies are IgG1 or IgG4.

An embodiment of the invention relates to an ADC wherein Ab is ahumanized antibody comprising variable domains VH and VL as abovedescribed in the format IgG1. More preferably, said humanized antibodycomprises a constant domain for the VH of sequence SEQ ID No. 43 and aKappa domain for the VL of sequence SEQ ID No. 45.

An embodiment of the invention relates to an ADC wherein Ab is ahumanized antibody comprising variable domains VH and VL as abovedescribed in the format IgG4. More preferably, said humanized antibodycomprises a constant domain for the VH of sequence SEQ ID No. 44 and aKappa domain for the VL of sequence SEQ ID No. 45.

Still another embodiment of the invention relates to an ADC wherein Abis an antibody selected from:

a) an antibody comprising a heavy chain of sequence SEQ ID No. 37 or anysequence exhibiting at least 80% identity with SEQ ID No. 37 and a lightchain of sequence SEQ ID No. 39 or any sequence exhibiting at least 80%identity with SEQ ID No. 39; and

b) an antibody comprising a heavy chain of sequence SEQ ID No. 38 or anysequence exhibiting at least 80% identity with SEQ ID No. 38 and a lightchain of sequence SEQ ID No. 40 or any sequence exhibiting at least 80%identity with SEQ ID No. 40.

For more clarity, the following table 6 illustrates non limitativeexamples of sequences of the VH and VL for the variant 1 (Var. 1) andthe variant 3 (Var. 3) of the humanized antibody hz208F2. It alsocomprises the consensus sequence for the variant 2 (Var. 2).

TABLE 6 Heavy Chain Light chain SEQ ID No. hz208F2 Variable domain (VH)33 (var. 1) Variable domain (VL) 35 Full length 37 Full length 39hz208F2 Variable domain (VH) 34 (Var. 3) Variable domain (VL) 36 Fulllength 38 Full length 40 hz208F2 Variable domain (VH) 41 (Var. 2)Variable domain (VL) 42

Another aspect of the present invention is an ADC wherein Ab is anantibody selected from i) an antibody produced by the hybridoma I-4757,I-4773, I-4775, I-4736 or I-4774 deposited at the CNCM, Institut PasteurFrance on the 30 May 2013, 26 Jun. 2013, 26 Jun. 2013, 24 Apr. 2013 and26 Jun. 2013, respectively, ii) an antibody which competes for bindingto IGF-1R with the antibody of i); or iii) an antibody which binds tothe same epitope of IGF-1R as does the antibody of i).

It is described herein a murine hybridoma selected from the hybridomaI-4757, 1-4773, I-4775, I-4736 and I-4774 deposited at the CNCM,Institut Pasteur France on the 30 May 2013, 26 Jun. 2013, 26 Jun. 2013,24 Apr. 2013 and 26 Jun. 2013, respectively.

It is also described an isolated nucleic acid coding for an antibody, orfor an antigen binding fragment thereof, according to the invention.

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”,“polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and“nucleotide sequence”, used interchangeably in the present description,mean a precise sequence of nucleotides, modified or not, defining afragment or a region of a nucleic acid, containing unnatural nucleotidesor not, and being either a double-strand DNA, a single-strand DNA ortranscription products of said DNAs.

The nucleic sequences of the antibodies of the ADC of present inventionhave been isolated and/or purified, i.e., they were sampled directly orindirectly, for example by a copy, their environment having been atleast partially modified. Isolated nucleic acids obtained by recombinantgenetics, by means, for example, of host cells, or obtained by chemicalsynthesis should also be mentioned here.

It is also described a vector comprising a nucleic acid coding for anantibody of the ADC of the invention, or for an antigen binding fragmentthereof, according to the invention.

It is disclosed notably targets cloning and/or expression vectors thatcontain such a nucleotide sequence.

The vectors preferably contain elements which allow the expressionand/or the secretion of nucleotide sequences in a given host cell. Thevector thus may contain a promoter, translation initiation andtermination signals, as well as suitable transcription regulationregions. It must be able to be maintained in a stable manner in the hostcell and may optionally have specific signals which specify secretion ofthe translated protein. These various elements are selected andoptimized by a person skilled in the art according to the host cellused. For this purpose, the nucleotide sequences can be inserted inself-replicating vectors within the chosen host or be integrativevectors of the chosen host.

The vectors are, for example, vectors of plasmid or viral origin. Theyare used to transform host cells in order to clone or express thenucleotide sequences of the invention.

Such vectors are prepared by methods typically used by a person skilledin the art and the resulting clones can be introduced into a suitablehost by standard methods such as lipofection, electroporation,conjugation, heat shock or chemical methods.

It is disclosed also isolated host cells transformed by or comprising avector as above described.

The host cell can be selected among prokaryotic or eukaryotic systemssuch as bacterial cells, for example, but also yeast cells or animalcells, notably mammal cells (with the exception of human). Insect orplant cells can also be used.

It is also disclosed a method for the production of an antibodyaccording to the invention, or an antigen binding fragment thereof,characterized in that said method comprises the following steps:

a) the culture in a medium with the suitable culture conditions for ahost cell as disclosed above; and

b) the recovery of the antibody thus produced from the culture medium orfrom said cultured cells.

The transformed cells are of use in methods for the preparation ofrecombinant antibodies. Methods for the preparation of antibodies inrecombinant form using a vector and/or a cell transformed by a vector,are also comprised in the present specification. Preferably, a celltransformed by a vector as above described is cultured under conditionsthat allow the expression of the aforesaid antibody and recovery of saidantibody.

As already mentioned, the host cell can be selected among prokaryotic oreukaryotic systems. In particular, it is possible to identify thenucleotide sequences that facilitate secretion in such a prokaryotic oreukaryotic system. A vector according to the invention carrying such asequence can thus be used advantageously for the production ofrecombinant proteins to be secreted. Indeed, the purification of theserecombinant proteins of interest will be facilitated by the fact thatthey are present in the supernatant of the cellular culture rather thaninside host cells.

The antibody of the ADC of the invention can also be prepared bychemical synthesis. One such method of preparation is also an object ofthe invention. A person skilled in the art knows methods for chemicalsynthesis, such as solid-phase techniques or partial solid-phasetechniques, by condensation of fragments or by conventional synthesis insolution. Polypeptides obtained by chemical synthesis and capable ofcontaining corresponding unnatural amino acids are also comprised in theinvention.

The ADC comprising antibody likely to be obtained by the method abovedescribed are also comprised in the present invention.

According to a particular aspect, the invention concerns an ADC whereinAb is an antibody, or an antigen binding fragment thereof, as abovedescribed for use as an addressing vehicle for delivering a cytotoxicagent at a host target site, said host target site consisting of anepitope localized into IGF-1R, preferably the IGF-1R extracellulardomain, more preferably the human IGF-1R (SEQ ID No. 50) and still morepreferably the human IGF-1R extracellular domain (SEQ ID No. 51), andstill more preferably to the N-terminal of the human IGF-1Rextracellular domain (SEQ ID No. 52), or any natural variant sequencethereof.

In a preferred embodiment, said host target site is a target site of amammalian cell, more preferably of a human cell, more preferably cellswhich naturally or by way of genetic recombination, express IGF-1R.

I.2: Axl Antibodies

Without any contradictory indication, the definition or expression usedand defined in the paragraph “I.1, IGF-1R antibodies” are the same inthe present paragraph and can be applied to all the ADC of the presentinvention.

The Axl antibody is capable of binding to the human protein Axl. Moreparticularly, the said target is an epitope located into theextracellular domain of Axl (referred as the Axl ECD for “Axl ExtraCellular Domain”).

The Axl ECD is a 451 amino acids fragment, corresponding to amino acids1-451 of the sequence SEQ ID No. 103, which sequence is represented inthe sequence listing as SEQ ID No. 105. Amino acids 1-25 correspondingto the signal peptide, the ECD of the human protein Axl without thesignal peptide corresponds to the amino acids 26-451 of the sequence SEQID No. 104, represented by the sequence SEQ ID No. 106.

The Axl antibody of the ADC of the invention comprises the three lightchain CDRs comprising the sequences SEQ ID Nos. 56, 57 and 58, or anysequence exhibiting at least 90%, preferably 95% and 98% identity withSEQ ID Nos. 56, 57 and 58; and the three heavy chain CDRs comprising thesequences SEQ ID Nos. 59, 60 and 61, or any sequence exhibiting at least90%, preferably 95% and 98% identity with SEQ ID Nos. 59, 60 and 61.

In an embodiment, the Axl antibody comprises the three light chain CDRscomprising respectively the sequences SEQ ID Nos. 56, 57 and 58; and thethree heavy chain CDRs comprising respectively the sequences SEQ ID Nos.59, 60 and 61.

In an embodiment, the Axl antibody consists of the m1613F12 comprisingi) a light chain variable domain of sequence SEQ ID No. 62, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 62; and/or ii) a heavy chain variable domain ofsequence SEQ ID No. 63, or any sequence exhibiting at least 80%,preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 63.

For more clarity, table 7a below summarizes the various amino acidsequences corresponding to the Axl antibody.

TABLE 7a CDR numbering Heavy chain Light chain SEQ ID NO. 1613F12 IMGT ®CDR-L1 56 CDR-L2 57 CDR-L3 58 CDR-H1 59 CDR-H2 60 CDR-H3 61 variabledomain 62 variable domain 63

In an embodiment, the Axl antibody consists of the c1613F12 comprisingthe three light chain CDRs comprising the sequences SEQ ID No. 56, 57and 58, or any sequence exhibiting at least 80%, preferably 85%, 90%,95% and 98% identity with SEQ ID No. 56, 57 and 58; and the three heavychain CDRs comprising the sequences SEQ ID No. 59, 60 and 61, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 59, 60 and 61.

In an embodiment, c1613F12 comprises the three light chain CDRscomprising respectively the sequences SEQ ID Nos. 56, 57 and 58; and thethree heavy chain CDRs comprising respectively the sequences SEQ ID Nos.59, 60 and 61.

In an embodiment, the Axl antibody consists of the c1613F12 comprisingi) a light chain variable domain of sequence SEQ ID No. 62, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 62; and/or ii) a heavy chain variable domain ofsequence SEQ ID No. 63, or any sequence exhibiting at least 80%,preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 63.

In an embodiment, the Axl antibody consists of the hz1613F12 comprisingthe three light chain CDRs comprising the sequences SEQ ID No. 56, 57and 58, or any sequence exhibiting at least 80%, preferably 85%, 90%,95% and 98% identity with SEQ ID No. 56, 57 and 58; and the three heavychain CDRs comprising the sequences SEQ ID No. 59, 60 and 61, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 59, 60 and 61.

In an embodiment, hz1613F12 comprises the three light chain CDRscomprising respectively the sequences SEQ ID Nos. 56, 57 and 58; and thethree heavy chain CDRs comprising respectively the sequences SEQ ID Nos.59, 60 and 61.

In an embodiment, hz1613F12 comprises a light chain variable domainconsisting of the sequence SEQ ID No. 70, or any sequence exhibiting atleast 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 70;and the three heavy chain CDRs consisting of sequences SEQ ID No. 59, 60and 61.

In another embodiment of the invention, hz1613F12 comprises a lightchain variable domain of sequence selected in the group consisting ofSEQ ID No. 71 to 81 or any sequence exhibiting at least 80%, preferably85%, 90%, 95% and 98% identity with SEQ ID No. 71 to 81; and the threeheavy chain CDRs consisting of SEQ ID No. 59, 60 and 61.

In order to illustrate the identity percentage as defined before, by“any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 70 to 81”, its is intended to designate thesequences exhibiting the three light chain CDRs SEQ ID No. 56, 57 and 58and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 70 to 81 outside thesequences corresponding to the CDRs (i.e. SEQ ID No. 56, 57 and 58).

For more clarity, table 7b below summarizes the various amino acidsequences corresponding to the humanized Axl antibody light chain (VL)of the ADC of the invention (with hz.=humanized)

TABLE 7b Version SEQ ID NO. hz1613F12 VL consensus 70 VL1 71 VL1 I2V 72VL1 M4I 73 VL2.1 74 VL2.1 V49T 75 VL2.1 P50N 76 VL2.2 77 VL2.2 V49T 78VL2.2 P50N 79 VL2.3 80 VL3 81

In an embodiment, hz1613F12 comprises a heavy chain variable domainconsisting of the sequence SEQ ID No. 82, or any sequence exhibiting atleast 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 82;and the three light chain CDRs consisting of sequences SEQ ID No. 56, 57and 58.

In another embodiment, hz1613F12 comprises a heavy chain variable domainof sequence selected in the group consisting of SEQ ID No. 83 to 102, orany sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 83 to 102; and the three light chain CDRsconsisting of SEQ ID No. 56, 57 and 58.

In order to illustrate the identity percentage as defined before, by“any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 82 to 102”, its is intended to designate thesequences exhibiting the three heavy chain CDRs SEQ ID No. 59, 60 and 61and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and98%, identity with the full sequence SEQ ID No. 82 to 102 outside thesequences corresponding to the CDRs (i.e. SEQ ID No. 59, 60 and 61).

For more clarity, table 7c below summarizes the various amino acidsequences corresponding to the humanized antigen binding protein heavychain (VH) of the invention (with hz.=humanized)

TABLE 7c Version SEQ ID NO. hz1613F12 VH consensus 82 VH1 83 VH1 M39I 84VH1 W55R N66K 85 VH1 I84S 86 VH1 S85N 87 VH1 I84N S85N 88 VH2.1 89 VH2.1Q3H 90 VH2.1 W55R 91 VH2.1 N66K 92 VH2.1 W55R N66K 93 VH2.1 R80S 94VH2.1 N66K R80S 95 VH2.2 96 VH2.2 M89L 97 VH2.3 98 VH2.3 W55R 99 VH2.3Q3H W55R 100 VH2.4 101 VH3 102

In an embodiment, hz1613F12 comprises a light chain variable domain ofsequence selected in the group consisting of SEQ ID No. 70 and anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 70; and a heavy chain variable domain ofsequence selected in the group consisting of SEQ ID No. 82 and anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 82.

In an embodiment, hz1613F12 comprises a light chain variable domain ofsequence selected in the group consisting of SEQ ID No. 71 to 81, or anysequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 71 to 81; and a heavy chain variable domain ofsequence selected in the group consisting of SEQ ID No. 83 to 102, orany sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID No. 83 to 102.

Another aspect of the present invention is an ADC wherein Ab is anantibody selected from i) an antibody produced by the hybridoma I-4505,deposited at the CNCM, Institut Pasteur France on the 28 Jul. 2011, ii)an antibody which competes for binding to Axl with the antibody of i);or iii) an antibody which binds to the same epitope of Axl as does theantibody of i).

According to another aspect, It is disclosed a murine hybridoma selectedfrom the hybridoma I-4505.

Table 8 below summarizes the nucleotide sequences concerning CDRs of the1613F12.

TABLE 8 CDR Antibody numbering Heavy chain Light chain SEQ ID NO.1613F12 IMGT ® CDR-L1 64 CDR-L2 65 CDR-L3 66 CDR-H1 67 CDR-H2 68 CDR-H369

In an embodiment, nucleic acid sequences of the CDRs can be selectedfrom the sequences SEQ ID Nos. 64 to 69.

A non limitative embodiment it is disclosed herein an isolated nucleicacid or a combination thereof coding for an antibody characterized inthat said nucleic acid or its combination comprises the 6 CDRs sequencesSEQ ID Nos. 64 to 69.

I.3: HER2 Antibodies

The HER2 antibodies can consist of any antibody capable of binding toHER2 such as, without limitation, TrasGEX (Glycotope), Trastuzumab,Pertuzumab or any biosimilar thereof.

In a preferred embodiment, the HER2 antibody is the Trastuzumab(Herceptin®; 4D5; Genentech, San Francisco, Calif.).

II—The Drug (D)

The drug moiety according to the invention has the following formula(II)

-   -   wherein:    -   R₁ is H or OH;    -   R₂ is a group: (C₁-C₆)alkyl (e.g. methyl), COOH,        COO—((C₁-C₆)alkyl) (such as COOMe) or thiazolyl (such as        thiazol-2-yl);    -   R₃ is H or a (C₁-C₆)alkyl group (such as methyl), in particular        a (C₁-C₆)alkyl group,    -   A is:        -   a group of formula -Het-Alk- wherein Alk is a            (C₁-C₈)alkanediyl group and is linked to NR₃, and Het is a            heterocycle optionally substituted by a (C₁-C₆)alkyl group            and containing at least one nitrogen atom, said nitrogen            atom being linked to L, or        -   a group of formula -A_(a)-A_(b)- wherein A_(a) is linked to            L and is O or NR₉ with R₉ being H or (C₁-C₆)alkyl (such as            methyl) and A_(b) is linked to NR₃ and is:            -   a (C₁-C₈)alkanediyl group (such as —(CH₂)_(m)— with m                comprised between 1 and 8),            -   a                —(CH₂CH₂X₁)_(a1)(CH₂CH₂X₂)_(a2)(CH₂CH₂X₃)_(a3)(CH₂CH₂X₄)_(a4)CH₂CH₂—                group with X₁, X₂, X₃ and X₄ each independently of one                another representing O or NR₈; a1, a2, a3 and a4 each                independently of one another representing 0 or 1 (in                particular with a1+a2+a3+a4=1 or 2, in particular 1);                and R₈ representing H or a (C₁-C₆)alkyl group (such as                methyl),            -   an aryl-(C₁-C₈)alkanediyl or                heterocycle-(C₁-C₈)alkanediyl group, said group being                optionally substituted by a (C₁-C₆)alkyl group, the aryl                or heterocycle moiety being linked to A_(a) and the                (C₁-C₈)alkanediyl moiety being linked to NR₃;

the wavy line indicates the point of attachment to L.

The radicals R₂, R₃ and A may be chiral groups and may be in the form oftheir different stereoisomers and optionally in the form of a mixture ofstereoisomers.

By <<stereoisomer>>, in the meaning of the present invention is meant ageometric isomer or an optical isomer.

Geometrical isomers result from the different position of thesubstituents on a double bond which may therefore have a Z or Econfiguration.

Optical isomers result in particular from the different position inspace of the substituents on a carbon atom comprising 4 differentsubstituents. This carbon atom then forms a chiral or asymmetric centre.Optical isomers comprise diastereoisomers and enantiomers. Opticalisomers which are images of one another in a mirror but which cannot besuperimposed are called <<enantiomers>>. Optical isomers which are notsuperimposable images of one another in a mirror are called<<diastereoisomers>>.

A mixture containing equal quantities of two individual enantiomer formsof opposite chirality is called a <<racemic mixture>>.

By <<alkyl>> in the present invention is meant a monovalent linear orbranched, saturated hydrocarbon chain. For example, mention can be madeof methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl or hexyl groups.

By <<(C_(x)-C_(y))alkyl>> in the meaning of the present invention ismeant an alkyl chain such as defined above comprising x to y carbonatoms. Therefore, a (C₁-C₆)alkyl group is an alkyl chain having 1 to 6carbon atoms. The (C₁-C₆)alkyl is advantageously a (C₁-C₄)alkyl,preferably a (C₁-C₂)alkyl.

By <<alkanediyl>> in the present invention is meant a divalent linear orbranched, saturated hydrocarbon chain. For example, mention can be madeof methanediyl, ethanediyl, propanediyl, butanediyl, pentanediyl,hexanediyl, and the like.

By <<(C_(x)-C_(y))alkanediyl>> in the meaning of the present inventionis meant an alkanediyl chain such as defined above comprising x to ycarbon atoms. Therefore, a (C₁-C₈)alkanediyl group is an alkanediylchain having 1 to 8 carbon atoms.

By <<aryl>> in the meaning of present invention is meant an aromatichydrocarbon group preferably having 6 to 10 carbon atoms and able tocomprise one or two fused rings. For example a phenyl or a naphthyl canbe cited. Advantageously it is a phenyl.

By <<heterocycle>> in the meaning of present invention is meant asaturated, unsaturated or aromatic hydrocarbon group having 1 or 2 fusedrings and in which one or more, advantageously 1 to 4, moreadvantageously 1 or 2 of the carbon atoms are each replaced by aheteroatom chosen from among oxygen, nitrogen and sulfur. Advantageouslythe heterocycle comprises 5 to 10 carbon atoms and heteroatoms. Forexample, mention can be made of furan, pyrrole, thiophene, thiazole,isothiazole, oxadiazole, imidazole, oxazole, isoxazole, pyridine,pyrimidine, piperazine, piperidine, quinazoline, quinoline, quinoxaline,benzofuran, benzothiophene, indoline, indolizine, benzothiazole,benzothiophene, benzopyran, benzoxazole, benzo[1,3]dioxole,benzoisoxazole, benzimidazole, chromane, chromene, dihydrobenzofuran,dihydrobenzothiophene, dihydroisoxazole, isoquinoline, dihydrobenzo[1,4]dioxin, imidazo[1,2-a]pyridine, furo[2,3-c]pyridine,2,3-dihydro-1H-indene, [1,3]dioxolo[4,5-c]pyridine,pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrimidine,tetrahydronaphthalene and benzo[b][1,4]oxazin.

In the present invention, the heterocycle is more particularly asaturated, unsaturated or aromatic ring with 5 to 6 members comprising 1or 2 nitrogen atoms. For example, mention can be made of pyrrole,imidazole, pyridine, pyrimidine, piperazine and piperidine rings.Preferably it is a pyridine, a piperidine, or an imidazole.

Among the drug moieties of the invention, one particularly appreciatedclass of drug moieties corresponds to the formula (II) drug moieties inwhich R₁ is OH and R₂ represents a (C₁-C₆)alkyl group, such as methyl.

Another particularly appreciated class of drug moieties corresponds tothe formula (II) drug moieties in which R₁ is a hydrogen and R₂ is athiazolyl (in particular a thiazol-2-yl group).

Another class of particularly appreciated drug moieties corresponds tothe formula (II) drug moieties in which R₁ is a hydrogen and R₂ is aCOO—((C₁-C₆)alkyl) group such as COOMe.

Another class of particularly appreciated compounds corresponds to theformula (II) drug moieties in which R₁ is a hydrogen and R₂ is a COOHgroup.

According to one particular embodiment of the present invention, R₂ is amethyl, COOH, COOMe or thiazol-2-yl group.

Therefore the drug moieties of the invention are advantageously formula(II) drug moieties in which:

-   -   R₁═OH and R₂=Me (methyl), or    -   R₁═H and R₂═COOH, COOMe or thiazol-2-yl.

According to a preferred embodiment, R₁═H and R₂═COOH, COOMe orthiazol-2-yl, preferably R₂═COOH or COOMe, and more preferably R₂═COOH.

R₃ particularly represents H or a methyl group, advantageously a methylgroup.

In a preferred embodiment, R₁ is H, R₂ is COOH and R₃ is a methyl group.

In the definition of A:

-   -   the (C₁-C₈)alkanediyl group is advantageously a        (C₁-C₆)alkanediyl group, preferably a (C₁-C₄)alkanediyl group        and more preferably a (C₁-C₂)alkanediyl group, and in particular        it is a straight chain having the formula —(CH₂)_(m)— wherein m        is an integer comprised between 1 and 8, advantageously between        1 and 6, preferably between 1 and 4, more preferably m is 1 or        2,    -   the aryl group is advantageously a phenyl group, and    -   the heterocycle is advantageously a saturated, unsaturated or        aromatic ring with 5 or 6 members having 1 or 2 nitrogen atoms;        for example, a pyrrole, imidazole, pyridine, pyrimidine,        piperazine, or piperidine ring; preferably a pyridine,        piperidine or imidazole; more preferably a pyridine.

When A is a group of formula -Het-Alk-, Het is more particularly aheterocycle chosen among a piperidine and an imidazole.

When A is a group of formula -A_(a)-A_(b)- wherein A_(b) is aheterocycle-(C₁-C₈)alkanediyl group optionally substituted by a(C₁-C₆)alkyl group, the heterocycle is more particularly a pyridine.

According to a preferred embodiment, A is a group of formula-A_(a)-A_(b)- as defined above.

A_(a) is O or NR₉, and preferably NR₉, with R₉ as defined above, andpreferably with R₉═H or Me.

According to a particular embodiment of the invention, A_(b) representsa group:

-   -   (C₁-C₆)alkanediyl, in particular (C₂-C₆)alkanediyl (such as        —(CH₂)_(m)— with m comprised between 1 and 6, preferably between        2 and 6),    -   —(CH₂CH₂X₁)_(a1)(CH₂CH₂X₂)_(a2)CH₂CH₂— with a1+a2 advantageously        representing 1 or 2, in particular 1,    -   aryl-(C₁-C₆)alkanediyl, or    -   heterocycle-(C₁-C₆)alkanediyl optionally substituted by a        (C₁-C₆)alkyl group (notably unsubstituted).

According to another particular embodiment of the invention, A_(b)represents a group:

-   -   (C₁-C₄)alkanediyl, in particular (C₂-C₄)alkanediyl (such as        —(CH₂)_(m)— with m comprised between 1 and 4 and preferably        being 2, 3 or 4),    -   —(CH₂CH₂X₁)CH₂CH₂— or —(CH₂CH₂X₁)(CH₂CH₂X₂)CH₂CH₂—,    -   aryl-(C₁-C₄)alkanediyl, or    -   heterocycle-(C₁-C₄)alkanediyl optionally substituted by a        (C₁-C₆)alkyl group (notably unsubstituted).

According to another particular embodiment of the invention, A_(b)represents a group:

-   -   (C₁-C₄)alkanediyl, in particular (C₂-C₄)alkanediyl (such as        —(CH₂)_(m)— with m comprised between 1 and 4 and preferably        being 2, 3 or 4),    -   —(CH₂CH₂X₁)CH₂CH₂—,    -   aryl-(C₁-C₂)alkanediyl, or    -   heterocycle-(C₁-C₂)alkanediyl optionally substituted by a        (C₁-C₆)alkyl group (notably unsubstituted).

According to another particular embodiment, A_(b) represents:

an aryl-(C₁-C₈)alkanediyl group; or a heterocycle-(C₁-C₈)alkanediylgroup optionally substituted by a (C₁-C₆)alkyl group (notablyunsubstituted);

notably an aryl-(C₁-C₆)alkanediyl group; or aheterocycle-(C₁-C₆)alkanediyl group optionally substituted by a(C₁-C₆)alkyl group (notably unsubstituted);

advantageously an aryl-(C₁-C₄)alkanediyl group; or aheterocycle-(C₁-C₄)alkanediyl group optionally substituted by a(C₁-C₆)alkyl group (notably unsubstituted);

and preferably an aryl-(C₁-C₂)alkanediyl group; or aheterocycle-(C₁-C₂)alkanediyl group optionally substituted by a(C₁-C₆)alkyl group (notably unsubstituted).

According to yet another particular embodiment, A_(b) is anaryl-(C₁-C₈)alkanediyl group; notably an aryl-(C₁-C₆)alkanediyl group;advantageously an aryl-(C₁-C₄)alkanediyl group; and preferably anaryl-(C₁-C₂)alkanediyl group.

In the above particular embodiments for A_(b), the aryl group isadvantageously a phenyl group.

In the above particular embodiments for A_(b), the heterocycle isadvantageously a saturated, unsaturated or aromatic ring with 5 or 6members having 1 or 2 nitrogen atoms. For example, mention can be madeof pyrrole, imidazole, pyridine, pyrimidine, piperazine, or piperidinerings. Preferably it is a pyridine, piperidine or imidazole, and morepreferably, it is a pyridine.

Advantageously, A_(b) represents a group:

-   -   phenyl-(C₁-C₂)alkanediyl, or    -   heterocycle-(C₁-C₂)alkanediyl optionally substituted by a        (C₁-C₆)alkyl group (notably unsubstituted), the heterocycle        being a saturated, unsaturated or aromatic ring with 5 or 6        members comprising 1 or 2 nitrogen atoms, chosen in particular        from among pyridine, piperidine and imidazole, and being        preferably a pyridine.

In a preferred embodiment, A_(b) is a phenyl-(C₁-C₂)alkanediyl group.

According to a preferred embodiment, A has the following formula:

and preferably the following formula:

wherein:

R₉ and m are as defined previously, and preferably with R₉=H or Me andm=1 or 2,

the wavy line indicates the point of attachment to L, and

the asterisk indicates the point of attachment to NR₃.

Advantageously, the drug moiety is chosen from among the followingmoieties:

Preparation of the Drug (of Formula DH):

The drug can be prepared using the general methods described in thefollowing synthesis schemes, optionally supplemented by any standardoperation when needed that is described in the literature or well knownto persons skilled in the art, or described in the examples in theexperimental part hereof.

Scheme 1 illustrates the first general method which can be used toprepare the drug. In the above general formulas, R₁, R₂ and R₃ are suchas previously defined, R₄ represents -AH, and R_(4a) represents a R₄group such as previously defined optionally in protected form and G is aprotective group.

The first step consists of the condensing of compound (II), protected onits amine function by a protective group G, with compound (III). X mayrepresent a leaving group such as a chlorine. In this case the firststep consists of the reaction between an acid chloride and an amine.This reaction can be conducted using methods and techniques well knownto those skilled in the art. In one particularly appreciated method, thetwo entities are caused to react in the presence of an organic orinorganic base e.g. Et₃N, iPr₂NEt, pyridine, NaH, Cs₂CO₃, K₂CO₃ in asolvent such as THF, dichloromethane, DMF, DMSO, at a temperaturenotably between −20° C. and 100° C. X may also be a hydroxyl (OH). Inthis case, the first step is a condensation reaction between thecarboxylic acid (II) and the amine (III). This reaction can be performedfollowing methods and techniques well known to skilled persons. In oneparticularly appreciated method, these two entities are caused to reactin the presence of a coupling agent such as1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC),3-hydroxy-1,2,3-benzotriazin-4(3H)-one, a tertiary amine such asdiisopropylethylamine, in a polar aprotic solvent such asdichloromethane or DMF, at a temperature notably between −15° C. and 40°C. In another particularly appreciated method, these two entities arecaused to react in the presence of diethyl phosphorocyanidate (DEPC), atertiary amine such as triethylamine, in a polar aprotic solvent such asdichloromethane or DMF, at a temperature of between −15° C. and 40° C.Another particularly appreciated method consists of causing these twoentities to react in the presence ofO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate(HATU), a tertiary amine such as diisopropylethylamine, in a polaraprotic solvent such as dichloromethane or DMF, at a temperature ofbetween −15° C. and 100° C.

After deprotection of the intermediate using techniques well known tothose skilled in the art (<<Protective Groups in Organic Synthesis>>, T.W. Greene, John Wiley & Sons, 2006 and <<Protecting Groups>>, P. J.Kocienski, Thieme Verlag, 1994), compound (IV) can be condensed withcompound (V) following the methods and techniques described above tolead to compound (VI) after a deprotection step. This compound can then,after condensation with the intermediate (VII) and optionaldeprotection, lead to the formation of the drug. Compound (VI) can alsobe coupled with a compound (VII′) in which R′₃ is a precursor of R₃, inparticular an R₃ group protected by a protective group. Couplingfollowed by deprotection of group R′₃ to lead to R₃ can be carried outfollowing the same procedures as described previously.

Scheme 2 illustrates the second general method which can be used toprepare the drug. In the above general formulas, G is a protectivegroup, R₁, R₂, R₃ and R_(4a) are such as previously defined, and R_(4b)represents -A_(c)-A_(a)H with A_(a) as defined previously and A_(c)representing:

-   -   a (C₁-C₇)alkanediyl group,    -   a        —(CH₂CH₂X₁)_(a1)(CH₂CH₂X₂)_(a2)(CH₂CH₂X₃)_(a3)(CH₂CH₂X₄)_(a4)CH₂—        group wherein the CH₂ group marked with an asterisk is linked to        the CHO group of R_(4b)CHO,    -   an aryl or heterocycle group,    -   an aryl-(C₁-C₇)alkanediyl or heterocycle-(C₁-C₇)alkanediyl        group, said group being optionally substituted by a (C₁-C₆)alkyl        group, the (C₁-C₇)alkanediyl moiety being linked to the CHO        group of R_(4b)CHO.

At the first step, compound (IX) protected on its amine function by aprotective group G is condensed with compound (VI). X may represent aleaving group e.g. a chlorine. In this case, the first step consists ofthe reaction between an acid chloride and an amine. This reaction can beperformed using methods and techniques well known to persons skilled inthe art. In one particularly appreciated method the two entities arecaused to react in the presence of an organic or inorganic base such asEt₃N, iPr₂NEt, pyridine, NaH, Cs₂CO₃, K₂CO₃ in a solvent such as THF,dichloromethane, DMF, DMSO at a temperature notably between −20° and100° C. X may also represent a hydroxyl. In this case, the first step isa condensation reaction between the carboxylic acid (IX) and the amine(VI). This reaction can be conducted following methods and techniqueswell known to skilled persons. In one particularly appreciated method,the two entities are caused to react in the presence of1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC),3-hydroxy-1,2,3-benzotriazin-4(3H)-one, a tertiary amine such asdiisopropylethylamine, in a polar aprotic solvent such asdichloromethane or DMF, at a temperature notably between −15° C. and 40°C. In another particularly appreciated method, these two entities arecaused to react in the presence of diethyl phosphorocyanidate (DEPC), atertiary amine such as triethylamine, in a polar aprotic solvent such asdichloromethane or DMF, at a temperature notably between −15° C. and 40°C.

After deprotection of the intermediate, using techniques well known toskilled persons, the obtained compound (VIII) can lead to the drug afterreaction with R₄Y. In this case, Y is a leaving group such as Cl, Br, I,OSO₂CH₃, OSO₂CF₃ or O-Tosyl. The reaction is conducted in the presenceof an organic or inorganic base such as Et₃N, iPr₂NEt, NaH, Cs₂CO₃,K₂CO₃, in a polar anhydrous solvent such as dichloromethane, THF, DMF,DMSO at a temperature notably between −20° and 100° C. In anotherparticularly appreciated method, compound (VIII) is caused to react withan aldehyde of formula R_(4b)—CHO where R_(4b) corresponds to aprecursor of R₄. In this case, the reaction is a reductive amination inthe presence of a reducing agent such as NaBH₄, NaBH₃CN, NaBH(OAc)₃, ina polar solvent such as 1,2-dichloroethane, dichloromethane, THF, DMF,MeOH, in the optional presence of titanium isopropoxide (IV), at a pHwhich can be controlled by the addition of an acid such as acetic acidat a temperature notably between −20° C. and 100° C.

In the foregoing synthesis schemes, a drug may lead to another drugafter an additional reaction step such as saponification for exampleusing methods well known to skilled persons whereby an R₂ grouprepresenting an ester (COOMe), is changed to an R₂ group representing acarboxylic acid (COOH).

If it is desired to isolate a drug containing at least one base functionin the state of an acid addition salt, this is possible by treating thefree base of the drug (containing at least one base function) with asuitable acid, preferably in equivalent quantity. The suitable acid mayin particular be trifluoroacetic acid.

III—the Linker (L)

“Linker”, “Linker Unit”, “L” or “link” means, in the present invention,a chemical moiety comprising a covalent bond or a chain of atoms thatcovalently attaches an antibody to at least one drug.

Linkers may be made using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation of cyctotoxicagents to the addressing system. Other cross-linker reagents may beBMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

The linker may be a “non cleavable” or “cleavable”.

In a preferred embodiment, it consists in a “cleavable linker”facilitating release of the drug in the cell. For example, anacid-labile linker, peptidase-sensitive linker, photolabile linker,dimethyl linker or disulfide-containing linker may be used. The linkeris, in a preferred embodiment, cleavable under intracellular conditions,such that cleavage of the linker releases the drug from the antibody inthe intracellular environment.

For example, in some embodiments, the linker is cleavable by a cleavingagent that is present in the intracellular environment (e.g., within alysosome or endosome or caveolea). The linker can be, for example, apeptidyl linker that is cleaved by an intracellular peptidase orprotease enzyme, including, but not limited to, a lysosomal or endosomalprotease. Typically, the peptidyl linker comprises at least twosuccessive amino acids or at least three successive amino acids or is atleast two amino acids long or at least three amino acids long. Cleavingagents can include cathepsins B and D and plasmin, all of which areknown to hydrolyze dipeptide drug derivatives resulting in the releaseof active drug inside target cells. For example, a peptidyl linker thatis cleavable by the thiol-dependent protease cathepsin-B, which ishighly expressed in cancerous tissue, can be used (e.g., a linkercomprising or being Phe-Leu or Gly-Phe-Leu-Gly). In specificembodiments, the peptidyl linker cleavable by an intracellular proteasecomprises or is Val-Cit or Phe-Lys. One advantage of using intracellularproteolytic release of the drug is that the drug is typically attenuatedwhen conjugated and the serum stabilities of the conjugates aretypically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker is hydrolyzable under acidic conditions. Forexample, an acid-labile linker that is hydrolyzable in the lysosome(e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconiticamide, orthoester, acetal, ketal, or the like) can be used. Such linkersare relatively stable under neutral pH conditions, such as those in theblood, but are unstable at below pH 5.5 or 5.0, the approximate pH ofthe lysosome. In certain embodiments, the hydrolyzable linker is athioether linker (such as, e.g., a thioether attached to the drug via anacylhydrazone bond).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

In certain preferred embodiments, the linker unit may have the followinggeneral formula:—(T)_(a)—(W)_(w)—(Y)_(y)—

wherein:

T is a stretcher unit;

a is 0 or 1;

W is an amino acid unit;

w is an integer ranging from 0 to 12;

Y is a spacer unit;

y is 0, 1 or 2.

The stretcher unit (T), when present, links the antibody to an aminoacid unit (W) when present, or to the spacer unit when present, ordirectly to the drug. Useful functional groups that can be present onthe antibody, either naturally or via chemical manipulation, includesulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of acarbohydrate, and carboxyl. Suitable functional groups are sulfhydryland amino. Sulfhydryl groups can be generated by reduction of theintramolecular disulfide bonds of the antibody, if present.Alternatively, sulfhydryl groups can be generated by reaction of anamino group of a lysine moiety of the antibody with 2-iminothiolane orother sulfhydryl generating reagents. In specific embodiments, theantibody is engineered to carry one or more lysines. More preferably,the antibody can be engineered to carry one or more Cysteines (cf.ThioMabs).

In certain specific embodiments, the stretcher unit forms a bond with asulfur atom of the antibody. The sulfur atom can be derived from asulfhydryl (—SH) group of a reduced antibody.

In certain other specific embodiments, the stretcher unit is linked tothe antibody via a disulfide bond between a sulfur atom of the antibodyand a sulfur atom of the stretcher unit.

In other specific embodiments, the reactive group of the stretchercontains a reactive site that can be reactive to an amino group of theantibody. The amino group can be that of an arginine or a lysine.Suitable amine reactive sites include, but are not limited to, activatedesters (such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters), anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates.

In yet another aspect, the reactive function of the stretcher contains areactive site that is reactive to a modified carbohydrate group that canbe present on the antibody. In a specific embodiment, the antibody isglycosylated enzymatically to provide a carbohydrate moiety or isnaturally glycosylated. The carbohydrate may be mildly oxidized with areagent such as sodium periodate and the resulting carbonyl unit of theoxidized carbohydrate can be condensed with a stretcher that contains afunctionality such as a hydrazide, an oxime, a reactive amine, ahydrazine, a thiosemicarbazide, a hydrazine carboxylate, or anarylhydrazide.

According to a particular embodiment, the stretcher unit has thefollowing formula:

whereinL₂ is (C₄-C₁₀)cycloalkyl-carbonyl, (C₂-C₆)alkyl or (C₂-C₆)alkyl-carbonyl(the cycloalkyl or alkyl moieties being linked to the nitrogen atom ofthe maleimide moiety), the asterisk indicates the point of attachment tothe amino acid unit, if present, to the spacer unit, if present, or tothe drug D, andthe wavy line indicates the point of attachment to the antibody Ab.

By “(C₄-C₁₀)cycloalkyl” in the present invention is meant a hydrocarboncycle having 4 to 10 carbon atoms including, but not limited to,cyclopentyl, cyclohexyl and the like.

L₂ can be advantageously (C₂-C₆)alkyl-carbonyl such as a pentyl-carbonylof the following formula:

wherein

the asterisk indicates the point of attachment to the amino acid unit,if present, to the spacer unit, if present, or to the drug D; and

the wavy line indicates the point of attachment to the nitrogen atom ofthe maleimide moiety.

The amino acid unit (W), when present, links the stretcher unit (T) ifpresent, or otherwise the antibody to the spacer unit (Y) if the spacerunit is present, or to the drug if the spacer unit is absent.

As above mentioned, (W)_(w) is absent (w=0) or may be a dipeptide,tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide,octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptideunit, wherein the amino acids forming the peptides can be different fromone another.

Thus (W)_(w) can be represented by the following formula:(W1)_(w1)(W2)_(w2)(W3)_(w3)(W4)_(w4)(W5)_(w5), wherein each W1 to W5represents, independently from one another, an amino acid unit and eachw1 to w5 is 0 or 1.

In some embodiments, the amino acid unit (W)_(w) may comprise amino acidresidues such as those occurring naturally, as well as minor amino acidsand non-naturally occurring amino acid analogs, such as citrulline.

The amino acid residues of the amino acid unit (W)_(w) include, withoutlimitation, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, lysine protected or not with acetylor formyl, arginine, arginine protected or not with tosyl or nitrogroups, histidine, ornithine, ornithine protected with acetyl or formyl,and citrulline. Exemplary amino acid linker components includepreferably a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide,notably a dipeptide or a tripeptide.

Exemplary dipeptides include: Val-Cit, Ala-Val, Ala-Ala, Val-Ala,Lys-Lys, Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp-Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-Nitro-Arg.

Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.

Exemplary tetrapeptide include: Gly-Phe-Leu-Gly (SEQ ID NO. 53),Ala-Leu-Ala-Leu (SEQ ID NO. 54).

Exemplary pentapeptide include: Pro-Val-Gly-Val-Val (SEQ ID NO. 55).

According to a particular embodiment, (W)_(w) can be a dipeptide (i.e.w=2) such as Val-Cit, or the linker lacks an amino acid unit (w=0). Whenthe linker lacks an amino acid unit, preferably it lacks also a spacerunit.

According to a preferred embodiment, w=0 (i.e. (W)_(w) is a single bond)or w=2 (i.e. (W)_(w) is a dipeptide) and (W)_(w) can thus be selectedfrom:

and in particular is Val-Cit,

wherein

the asterisk indicates the point of attachment to the spacer unit ifpresent, or to the drug D; and

the wavy line indicates the point of attachment to L₂.

Amino acid linker components can be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme, for example,a tumor-associated protease, cathepsin B, C and D, or a plasminprotease.

The amino acid unit of the linker can be enzymatically cleaved by anenzyme including, but not limited to, a tumor-associated protease toliberate the drug.

The amino acid unit can be designed and optimized in its selectivity forenzymatic cleavage by a particular tumor-associated protease. Thesuitable units are those whose cleavage is catalyzed by the proteases,cathepsin B, C and D, and plasmin.

The spacer unit (Y), when present, links an amino acid unit if present,or the stretcher unit if present, or otherwise the antibody to the drug.Spacer units are of two general types: self-immolative and nonself-immolative. A non self-immolative spacer unit is one in which partor all of the spacer unit remains bound to the drug after enzymaticcleavage of an amino acid unit from the antibody-drug conjugate.Examples of a non self-immolative spacer unit include, but are notlimited to a (glycine-glycine) spacer unit and a glycine spacer unit. Toliberate the drug, an independent hydrolysis reaction should take placewithin the target cell to cleave the glycine-drug unit bond.

In a particular embodiment, a non self-immolative the spacer unit (Y) isGly.

Alternatively, an antibody-drug conjugate containing a self-immolativespacer unit can release the drug without the need for a separatehydrolysis step. In these embodiments, (Y) is a residue of p-aminobenzylalcohol (PAB) unit that is linked to (W)_(w) via the nitrogen atom ofthe PAB group, and connected directly to the drug via a ester,carbonate, carbamate or ether group.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically equivalent to the PABgroup such as residues of 2-aminoimidazol-5-methanol derivatives andortho or para-aminobenzylacetals. Spacers can be used that undergofacile cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides, appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems and2-aminophenylpropionic acid amides.

In an alternate embodiment, the spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporateadditional drugs.

In a particular embodiment, the spacer unit (Y) is PAB-carbonyl with PABbeing

(the oxygen of the PAB unit being linked to the carbonyl), and y=1 orthe linker lacks a spacer unit (y=0).

In a particular embodiment, the linker has the following formula (III):

wherein

L₂ is (C₄-C₁₀)cycloalkyl-carbonyl, (C₂-C₆)alkyl or (C₂-C₆)alkyl-carbonyl(the carbonyl of these moieties, when present, being linked to (W)_(w)),

W represents an amino acid unit, with w representing an integercomprised between 0 and 5,

Y is PAB-carbonyl, with PAB being

(the oxygen of the PAB unit being linked to the carbonyl), and y is 0 or1 (preferably y is 0 when w is 0 and y is 0 or 1 when w is comprisedbetween 1 and 5),

the asterisk indicates the point of attachment to the drug D, and

the wavy line indicates the point of attachment to the antibody Ab.

Advantageously, L₂ is (C₂-C₆)alkyl-carbonyl such as a pentyl-carbonyl ofthe following formula:

wherein

the asterisk indicates the point of attachment to (W)_(w); and

the wavy line indicates the point of attachment to the nitrogen atom ofthe maleimide moiety.

According to a preferred embodiment, the linker L is selected from:

wherein the asterisk indicates the point of attachment to the drug D,and the wavy line indicates the point of attachment to the antibody Ab.

IV—the Antibody-Drug-Conjugate (ADC)

In a preferred embodiment, the antibody-drug conjugate of the inventionmay be prepared by any method known by the person skilled in the artsuch as, without limitation, i) reaction of a nucleophilic group of theantibody with a bivalent linker reagent followed by reaction with anucleophilic group of the drug or ii) reaction of a nucleophilic groupof the drug with a bivalent linker reagent followed by reaction with anucleophilic group of the antibody.

Nucleophilic groups on antibody include, without limitation, N-terminalamine groups, side chain amine groups (e.g. lysine), side chain thiolgroups, and sugar hydroxyl or amino groups when the antibody isglycosylated.

Nucleophilic groups on the drug include, without limitation, amine,thiol, and hydroxyl groups, and preferably amine groups.

Amine, thiol, and hydroxyl groups are nucleophilic and capable ofreacting to form covalent bonds with electrophilic groups on linkermoieties and linker reagents including, without limitation, activeesters such as NHS esters, HOBt esters, haloformates, and acid halides;alkyl and benzyl halides such as haloacetamides; aldehydes; ketones;carboxyl; and maleimide groups. The antibody may have reducibleinterchain disulfides, i.e. cysteine bridges. The antibody may be madereactive for conjugation with linker reagents by treatment with areducing agent such as DTT (dithiothreitol). Each cysteine bridge willthus form, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into the antibody through anyreaction known by the person skilled in the art. As non limitativeexample, reactive thiol groups may be introduced into the antibody byintroducing one or more cysteine residues.

Antibody-drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent. The sugars ofglycosylated antibody may be oxidized to form aldehyde or ketone groupswhich may react with the amine group of linker reagents or drug. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced to form stable amine linkages. In one embodiment, reaction ofthe carbohydrate portion of a glycosylated antibody with eithergalactose oxidase or sodium meta-periodate may yield carbonyl (aldehydeand ketone) groups in the protein that can react with appropriate groupson the drug. In another embodiment, proteins containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid.

In a preferred embodiment, the antibody-drug conjugate of the inventionis prepared by preparation of the drug-linker moiety followed bycoupling between a nucleophilic group of the antibody (for ex. the SHgroup of a cysteine moiety) and an electrophilic group of thedrug-linker moiety (for ex. a maleimide).

1. Drug-Linker

The Drug-Linker moiety can be prepared by coupling:

-   -   the linker with the drug,    -   a part of the linker with the drug before completing the        synthesis of the linker,    -   the linker with a part or a precursor of the drug before        completing the synthesis of the drug, or    -   a part of the linker with a part or a precursor of the drug        before completing the synthesis of the linker and the drug.

The coupling reactions are well known reactions for the one skilled inthe art between a nucleophilic group and an electrophilic group.

The nucleophilic group can be in particular an amine, thiol or hydroxylgroup, and notably an amine or hydroxyl group. In a preferred embodimentit is a primary or secondary amine group.

The electrophilic group can be a carboxylic acid group (COOH) optionallyin an activated form or an activated carbonate ester moiety.

By “activated form” of a carboxylic acid is meant a carboxylic acid inwhich the OH moiety of the COOH function has been replaced with anactivated leaving group (LG) enabling coupling of the activatedcarboxylic acid group with an amino group in order to form an amide bondand release the compound LG-H. Activated forms may be activated esters,activated amides, anhydrides or acyl halides such as acyl chlorides.Activated esters include derivatives formed by reaction of thecarboxylic acid group with N-hydroxybenzotriazole orN-hydroxysuccinimide.

By “activated carbonate ester” is meant a carbonate ester comprising a—OC(O)OR moiety in which OR represents a good leaving group enablingcoupling of the activated carbonate ester with an amino group in orderto form a carbamate moiety and release the compound ROH. The R group ofthe activated carbonate ester includes, without limitation, thep-nitro-phenyl, pentafluorophenyl, 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yland benzyl groups, preferably the p-nitro-phenyl and pentafluorophenylgroups.

When the linker has the following formula (III):

the Drug-Linker moiety has the following formula (IV):

and the last step of the synthesis of the Drug-Linker moiety isgenerally the coupling between a compound of the following formula (V):

where L₂ is as defined previously and LG represents a leaving groupnotably a halide such as a chloride or a group derived fromN-hydroxysuccinimide, and a compound of the following formula (VI):H—(W)_(w)—(Y)_(y)—D   (VI).

When y=1 and Y=PAB-carbonyl, the compound of formula (VI) can beprepared by the coupling between the drug (DH) and a compound of thefollowing formula (VII) or a protected form thereof:H—(W)_(w)—PAB—CO—OR   (VII)where W and w are as defined previously and R is as defined in thedefinition of the “activated carbonate ester”.

When the compound of formula (VII) is in a protected form, final step ofdeprotection is necessary.

When y=0, the compound (VI) has the formula H—(W)_(w)—D, wherein (W)_(w)and D are composed of amino acid units. Consequently, the compound (VI)can be prepared in this case by a conventional peptide synthesis methodwell known to the one skilled in the art.

2. Ab-Linker-Drug

A preferred embodiment according to the invention consists of a couplingbetween a cysteine present on the antibody and an electrophilic group ofthe Drug-Linker moiety, preferably with a maleimide moiety present onthe Drug-Linker moiety.

The maleimide-cysteine coupling can be performed by methods well knownto the person skilled in the art.

Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which can be linked to a drug moiety. Mostcysteine thiol residues in antibodies exist as disulfide bridges andmust be reduced with a reducing agent such as dithiothreitol (DTT) orTCEP, under partial or total reducing conditions. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of drug-linkerintermediate (D-L) or linker reagent relative to antibody, (ii) limitingthe conjugation reaction time or temperature, and (iii) partial orlimited reducing conditions for cysteine thiol modification.

The disulfide bond structure of human IgGs is now well established(reviewed in Liu and May, mAbs 4 (2012): 17-23). There are in fact manysimilarities and some differences with regard to the disulfide bondstructures of the 4 human IgG subclasses, namely IgG1, IgG2, IgG3 andIgG4. All IgG subclasses contain invariably 12 intra-chain disulfidebridges and the differences reside in their inter-chain disulfide bondsformed between heavy and light chains. Each intra-chain disulfide bondis associated with an individual IgG domain, i.e. variable (VL and VH)and constant (CL, CH1, CH2 and CH3) domains. The 2 heavy chains arelinked in their hinge region by a variable number of disulfide bridges:2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. The heavy and lightchains of the IgG1 are connected by a disulfide bond between the lastcysteine residue of the light chain and the fifth residue of the heavychain, whereas for the other subclasses, IgG2, IgG3 and IgG4, the lightchain is linked to the heavy chain by a disulfide bond between the lastcysteine residue of the light chain and the third cysteine residue ofthe heavy chain, which is located at the interface of VH and CH1domains. Disulfide bond structures other than these classical structureshave been described for IgG2 and IgG4 (reviewed in Liu and May, mAbs 4(2012): 17-23). Inter-chain disulfide bonds are highly solvent exposedand are consequently much more reactive than the intra-chain disulfidebonds, which are buried in anti-parallel beta-sheet structures withineach domain and are not solvent exposed. For these reasons, whatever theantibody isotype, coupling will take place on inter-chain exposedcysteine residues after mild reduction. Each inter-chain disulfidebridge can thus form, theoretically, two sites of conjugation.

Additional nucleophilic groups can be introduced into antibodies throughthe reaction of lysines with 2-iminothiolane (Traut's reagent) resultingin the conversion of an amine into a thiol. Reactive thiol groups mayalso be introduced into the antibody (or fragment thereof) byengineering one, two, three, four, or more cysteine residues (e.g.,preparing mutant antibodies comprising one or more non-native cysteineamino acid residues). U.S. Pat. No. 7,521,541 teaches engineeringantibodies by introduction of reactive cysteine amino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No.7,723,485; WO2009/052249). The engineered cysteine thiols may react withlinker reagents or the drug-linker reagents of the present inventionwhich have thiol-reactive, electrophilic groups such as maleimide oralpha-halo amides to form ADC with cysteine engineered antibodies andthe drug moieties. The location of the drug moiety can thus be designed,controlled, and known. The drug loading can be controlled since theengineered cysteine thiol groups typically react with thiol-reactivelinker reagents or drug-linker reagents in high yield. Engineering anIgG antibody to introduce a cysteine amino acid by substitution at asingle site on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A drug loading near 2 can be achieved with nearhomogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

For some antibody-drug conjugates, drug ratio may be limited by thenumber of attachment sites on the antibody. High drug loading, e.g. drugratio >5, may cause aggregation, insolubility, toxicity, or loss ofcellular permeability of certain antibody-drug conjugates. Typically,less drug moieties than the theoretical maximum are conjugated to anantibody during a conjugation reaction.

The drug loading also referred as the Drug-Antibody ratio (DAR) is theaverage number of drugs per cell binding agent.

In the case of antibody IgG1 and IgG4 isotypes, where the drugs arebound to cysteines after partial antibody reduction, drug loading mayrange from 1 to 8 drugs (D) per antibody, i.e. where 1, 2, 3, 4, 5, 6,7, and 8 drug moieties are covalently attached to the antibody.

In the case of an antibody IgG2 isotype, where the drugs are bound tocysteines after partial antibody reduction, drug loading may range from1 to 12 drugs (D) per antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 and 12 drug moieties are covalently attached to the antibody.

Compositions of ADC include collections of cell binding agents, e.g.antibodies, conjugated with a range of drugs, from 1 to 8 or 1 to 12.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectrometry, ELISA assay, andelectrophoresis.

As non limitative embodiment, it is presented herein the conjugationwith the IGF-1R antibody c208F2. In this case, the drug is coupled to atleast one cysteine selected from i) for the light chain of sequence SEQID No. 28, the residue Cys. in position 214 and ii) for the heavy chainof sequence SEQ ID No. 23, the residues Cys. in position 223, 229 and232.

As non limitative embodiment, it is presented herein the conjugationwith the IGF-1R antibody c208F2. In this case, the drug is coupled totwo, three or four, cysteines selected from i) for the light chain ofsequence SEQ ID No. 28, the residue Cys. in position 214 and ii) for theheavy chain of sequence SEQ ID No. 23, the residues Cys. in position223, 229 and 232

As non limitative embodiment, it is presented herein the conjugationwith the IGF-1R antibody hz208F2 (var. 1). In this case, the drug iscoupled to at least one cysteine selected from i) for the light chain ofsequence SEQ ID No. 39, the residue Cys. in position 214 and ii) for theheavy chain of sequence SEQ ID No. 37, the residues Cys. in position223, 229 and 232.

As non limitative embodiment, it is presented herein the conjugationwith the IGF-1R antibody hz208F2 (var. 3). In this case, the drug iscoupled to two, three or four, cysteines selected from i) for the lightchain of sequence SEQ ID No. 40, the residue Cys. in position 214 andii) for the heavy chain of sequence SEQ ID No. 38, the residues Cys. inposition 223, 229 and 232.

The same approach could be easily adapted to other antibodies such as,for example, the Axl antibodies.

An alternative consists of lysine coupling. An antibody may contain, forexample, many lysine residues that do not react with the drug-linkerintermediate (D-L) or linker reagent. Only the most reactive lysinegroups may react with an amine-reactive linker reagent. Also, only themost reactive cysteine thiol groups may react with a thiol-reactivelinker reagent.

Where the compounds of the invention are bound to lysines, drug loadingmay range from 1 to 80 drugs (D) per cell antibody, although an upperlimit of 40, 20, 10 or 8 may be preferred. Compositions of ADC includecollections of cell binding agents, e.g. antibodies, conjugated with arange of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The ADC of formula (I) according to the invention can be in the form ofa pharmaceutically acceptable salt.

In the present invention by “pharmaceutically acceptable” is meant thatwhich can be used in the preparation of a pharmaceutical compositionwhich is generally, safe non-toxic and neither biologically norotherwise undesirable, and which is acceptable for veterinary use aswell as for human pharmaceutical use.

By “pharmaceutically acceptable salt” of a compound is meant a saltwhich is pharmaceutically acceptable as defined herein and which has thedesired pharmacological activity of the parent compound.

Pharmaceutically acceptable salts notably comprise:

(1) the addition salts of a pharmaceutically acceptable acid formed withpharmaceutically acceptable inorganic acids such as hydrochloric,hydrobromic, phosphoric, sulfuric and similar acids; or formed withpharmaceutically acceptable organic acids such as acetic,trifluoroacetic, propionic, succinic, fumaric, malic, tartaric, citric,ascorbic, maleic, glutamic, benzoic, salicylic, toluenesulfonic,methanesulfonic, stearic, lactic and similar acids; and

(2) the addition salts of a pharmaceutically acceptable base formed whenan acid proton present in the parent compound is either replaced by ametallic ion e.g. an alkaline metal ion, an alkaline-earth metal ion oran aluminium ion; or coordinated with a pharmaceutically acceptableorganic base such as lysine, arginine and similar; or with apharmaceutically acceptable inorganic base such as sodium hydroxide,potash, calcium hydroxide and similar.

These salts can be prepared from the compounds of the inventioncontaining a base or acid function, and the corresponding acids or basesusing conventional chemical methods.

V—Treatment

Finally, the invention relates to an ADC as above described for use as amedicament, in particular in the treatment of cancer.

A further subject of the present invention is a formal (I) compound suchas defined above for use as medicinal product, in particular for thetreatment of cancer.

The present invention also concerns the use of a formula (I) compoundsuch as defined above for producing a medicinal product, particularlyintended for the treatment of cancer.

The present invention also concerns a method for treating cancercomprising the administration to a person in need thereof of aneffective mount of a formula (I) compound such as defined above.

Cancers can be preferably selected through Target-related cancersincluding tumoral cells expressing or over-expressing whole or part ofthe Target at their surface.

More particularly, said cancers are breast cancer, colon cancer,esophageal carcinoma, hepatocellular cancer, gastric cancer, glioma,lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer,rhabdomyosarcoma, renal cancer, thyroid cancer, uterine endometrialcancer, schwannoma, neuroblastoma, oral squamous cancer, mesothelioma,leiomyosarcoma, Kaposi sarcoma, acute leukemia, colorectal carcinoma,melanoma, pancreatic ductal adenocarcinoma and any drug resistancephenomena or cancers.

For the avoidance of doubt, by drug resistance Target-expressingcancers, it must be understood not only resistant cancers whichinitially express the Target but also cancers which initially do notexpress or overexpress the Target but which express the Target once theyhave become resistant to a previous treatment.

Another object of the invention is a pharmaceutical compositioncomprising the ADC as described in the specification.

More particularly, the invention relates to a pharmaceutical compositioncomprising the ADC of the invention with at least an excipient and/or apharmaceutical acceptable vehicle.

In the present description, the expression “pharmaceutically acceptablevehicle” or “excipient” is intended to indicate a compound or acombination of compounds entering into a pharmaceutical composition notprovoking secondary reactions and which allows, for example,facilitation of the administration of the active compound(s), anincrease in its lifespan and/or in its efficacy in the body, an increasein its solubility in solution or else an improvement in itsconservation. These pharmaceutically acceptable vehicles and excipientsare well known and will be adapted by the person skilled in the art as afunction of the nature and of the mode of administration of the activecompound(s) chosen.

The active ingredient can be administered in unit forms ofadministration, in a mixture with conventional pharmaceutical carriers,to animals or to human beings. Suitable unit forms of administrationcomprise forms via oral route and forms for administration viaparenteral route (subcutaneous, intradermal, intramuscular orintravenous).

As solid compositions, for oral administration, use can be made oftablets, pills, powders (hard or soft gelatine capsules) or granules. Inthese compositions, the active ingredient of the invention is mixed withone or more inert diluents such as starch, cellulose, sucrose, lactoseor silica, in a stream of argon. These compositions may also comprisesubstances other than diluents, for example one or more lubricants suchas magnesium stearate or talc, a colouring agent, a coating (coatedtablets) or a varnish.

The sterile compositions for parenteral administration may preferably beaqueous or non-aqueous solutions, suspensions or emulsions. As solventor vehicle, use can be made of water, propylene glycol, a polyethyleneglycol, vegetable oils, in particular olive oil, injectable organicesters e.g. ethyl oleate or other suitable organic solvents. Thesecompositions may also contain adjuvants, in particular wetting,isotonic, emulsifying, dispersing and stabilising agents. Sterilisationcan be performed in several manners, for example by sanitisingfiltration, by incorporating sterilising agents into the composition, byradiation or by heating. They can also be prepared in the form of solidsterile compositions which can be dissolved at the time of use insterile water or any other injectable sterile medium.

Preferably, these ADCs will be administered by the systemic route, inparticular by the intravenous route, by the intramuscular, intradermal,intraperitoneal or subcutaneous route, or by the oral route. In a morepreferred manner, the composition comprising the ADCs according to theinvention will be administered several times, in a sequential manner.

The invention concerns thus also a kit comprising at least i) anantibody-drug-conjugate according to the invention and/or apharmaceutical composition according to the invention and ii) a syringeor vial or ampoule in which the said antibody-drug-conjugate and/orpharmaceutical composition is disposed.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figures whoselegends are represented below.

FIGURE LEGENDS

FIGS. 1A-1C: Antibody binding to the human native IGF-1R by FACSanalyses. FIG. 1A represents the titration curve, on MCF-7 cell line.MFI represents the mean of fluorescent intensity. FIG. 1B represents theEC₅₀ of both murine and chimeric anti-IGF-1R antibodies on the MCF-7cell line. FIG. 1C represents the B_(max) of chimeric anti-IGF-1Rantibodies on MCF-7 cell line.

FIGS. 2A-2B: Evaluation of hIGF-1R recognition using transfected vs nontransfected cells. FIG. 2A) Represents titration curves of one chimericanti-IGF-1R Ab on IGF-1R⁺ cell line. MFI represents the mean offluorescent intensity. FIG. 2B represents the binding of chimericanti-IGF-1R Abs on the human IGF-1R⁻ cell line.

FIGS. 3A-3B: Evaluation of the specificity of Abs to IGF-1R vs hIR usingtransfected cells. FIG. 3A represents the binding of murine anti-IGF-1RAb on the hIR⁺ transfected cell line. FIG. 3B represents the binding ofchimeric anti-IGF-1R Ab on the IR+ cell line. MFI represents the mean offluorescent intensity. GRO5 anti-hIR Mab (Calbiochem) was introduced asa positive control.

FIG. 4: Binding of murine anti-IGF-1R Ab on the IM-9 cell line. MFIrepresents the mean of fluorescent intensity. GRO5 anti-hIR Mab wasintroduced as a positive control.

FIGS. 5A-5C: Evaluation of recognition of the monkey IGF-1R. FIG. 5Arepresents the titration curves of chimeric anti-IGF-1R Ab on the COS-7cell line. MFI represents the mean of fluorescent intensity. FIG. 5Brepresents the EC₅₀ of both murine and chimeric anti-IGF-1R antibodieson COS-7 cell line. FIG. 5C represents the EC₅₀ of chimeric anti-IGF-1Rantibodies on both NIH 3T3 transfected cells hIGF1R+ and COS-7 celllines.

FIG. 6: Sensorgrams obtained on a SPR technology based Biacore X100using a CM5 sensorchip activated with more the 11000 RU of mouseanti-Tag His antibody chemically grafted to the carboxymethyl dextranmatrix. The experiment was run at a flow rate of 30 μl/min at 25° C.using the HBS-EP+ as the running and samples diluting buffer. The figureshowed the superposition of 4 independent sensorgrams aligned on thex-axis at the beginning of the first injection of the analytes and onthe y-axis by the baseline defined just before this first injection. Thesensorgrams obtained with the capture of the human based sequence of therecombinant soluble IGF1R are marked by diamonds. The sensorgramsobtained with the capture of the cynomolgus based sequence of therecombinant soluble IGF1R are marked by triangles. White symbolscorrespond to the blank cycles (5 injections of the running buffer) andblack symbols correspond to the injections of the growing range ofconcentrations of c208F2 (5, 10, 20, 40 and 80 nM).

FIG. 7: Evaluation of the intrinsic effect of anti-hIGF-1R antibodies onthe receptor phosphorylation compared to IGF1.

FIG. 8: Inhibition of IGF-1R phosphorylation in response to IGF-1 bymurine anti-hIGF-1R

FIG. 9: Cell surface signal intensity of anti-IGF-1R antibodies isdown-regulated after cell incubation at 37° C. MCF-7 cells wereincubated at 4° C. or 37° C. for 4 h with 10 μg/ml of Abs. The figurerepresents the ΔMFI.

FIGS. 10A-10B: Antibody surface decay. Cell surface bound antibody wasassessed after 10, 20, 30, 60 and 120 min at 37° C. FIG. 10A representsthe % of remaining IGF-1R in comparison to the signal intensity measuredat 4° C. FIG. 10B represents Half Life calculation using Prims Softwareand using exponential decay fitting.

FIG. 11: Anti-hIGF-1R Abs are internalized. Cells were incubated with 10μg/ml of murine Abs for 0, 30 or 60 min at 37° C. cells werepermeabilized or not and incubated with a secondary anti-mouse IgG-Alexa488. Membrane corresponds to the signal intensity w/o permeabilization.Total correspond to the signal intensity after cell permeabilization andcytoplasmic corresponds to internalized Ab. The name of each evaluatedantibody is depicted on the top of each graph.

FIGS. 12A-12B: Imaging Ab internalization. FIG. 12A: MCF-7 cellsincubated with m208F2 for 20 min. at 4° C. and washed before incubation(W) at 37° C. for 15 (X), 30 (Y) and 60 (Z) min. Cells were fixed andpermeabilized. The m208F2 Ab was revealed using an anti-mouse IgGAlexa488 and Lamp-1 using a rabbit anti-Lamp-1 antibody and with asecondary anti-rabbit IgG Alexa 555. FIG. 12B: MCF-7 cells wereincubated for 30 minutes at 37° C. with anti-hIGF-1R murine antibodiesand stained as described above. Colocalization was identified using thecolocalization highliter plug-in of the ImageJ software.

FIG. 13: Involvement of the lysosome pathway in antibody degradation

FIG. 14: Acidic pH decreases binding capacity of the five murineanti-IGF-1R antibodies.

FIGS. 15A-15D: Binding characteristic of the first humanized form of thec208F2 Mab. Binding properties of the hz208F2 VH3/VL3 mAb was evaluatedon the human cell line MCF-7 (A), on the monkey cell line COS-7 (B) andon the transfected murine cell line expressing the human insulinreceptor (C). The binding of both the murine and the chimeric 208F2 mAbswas evaluated in parallel. The anti-hIR antibody clone GRO5 was used toverify the expression of the hIR on the transfixed cell line (D).

FIG. 16: hz208F2 VH3/VL3 antibody surface decay

FIG. 17: Superposition of to sensorgrammes obtained with a SPR basedBiacore X100 device at a temperature of 25° C. with a CM5 sensor chipactivated on both flowcells with around 12.000 RU of a mouse anti-TagHismonoclonal antibodies chemically grafted to the carboxymethyldextranmatrix using a HBS-EP+ as the running buffer at a flow rate of 30μl/min. Each sensorgrammes (the first one marked by triangles and thesecond one marked by diamonds) correspond to a complete cycle:

-   -   1—Injection during one minute of a solution of recombinant        h-IGF1R (10 μg/ml) on the second flowcell.    -   2—For the first sensorgramme: 5 injections of running buffer        during 90 s each    -   For the second sensorgramme: five injections in the growing        range of concentrations of the anti-IGF1R c208F2 antibody        solutions during 90 s each.    -   3—A delay of 300 s for the determination of the dissociation        kinetic rates.    -   4—A regeneration of the surface by an injection during 45 s of a        10 mM Glycine, HCl pH 1.5 buffer.

FIG. 18: The sensorgramme corresponding to the subtraction of the blanksensorgramme (5 injections of HBS-EP+) to the sensorgramme obtained withthe growing range of concentrations of the anti-IGF1R c208F2 solutionsis presented in grey. The theoretical sensorgramme corresponding to the1:1 model with the following parameters: k_(on)=(1.206±0.036)×10⁶M⁻¹·s⁻¹, k_(off)=(7.81±0.18)×10⁻⁵ s⁻¹, Rmax=307.6±0.3 RU is presented bya thin black line. The calculated concentrations of c208F2 are reportedon the graph: only the highest concentration (24 nM) is considered as aconstant).

FIG. 19: The dissociation constants correspond to the mean of the fourexperiments run for each antibody and correspond to the ratio:k_(off)/k_(on)×10¹² to be express in the pM unit. The error barscorrespond to the standard error (n=4).

FIG. 20: the half-lives correspond to the mean of the four experimentsrun for each antibody and correspond to the ratio: Ln(2)/k_(off)/3600 tobe express in the h unit.

The error bars correspond to the standard error (n=4).

FIG. 21: Cell cytotoxicity of anti-IGF-1R coupled with three differentcompounds. Five chimeric antibodies anti-IGF-1R were coupled with eitherE-13, G-13 or F-63. An irrelevant antibody c9G4 was also coupled withthe same compounds.

FIGS. 22A-22C: in vivo evaluation of c208F2-E-13 (FIG. 22A), c208F2-G-13(FIG. 22B) and c208F2-F-63 (FIG. 22C) in the MCF-7 xenograft model.

FIGS. 23A-23B: in vivo evaluation of both c208F2-E-13 (FIG. 23A) andc208F2-G-13 (FIG. 23B) compared to ADCs control (c9G4-E13 and c9G4-G-13)in the MCF-7 xenograft model.

FIGS. 24A, 24B and 24C: Binding specificity of 1613F12 on theimmobilized rhAxl-Fc protein (24A), rhDtk-Fc (24B) or rhMer-Fc (24C)proteins by ELISA.

FIG. 25: FACS analysis of the 1613F12 binding on human tumor cells

FIG. 26: ELISA experiments studying binding on rhAxl-Fc protein of bothm1613F12 and hz1613F12.

FIGS. 27A, 27B and 27C: Immunofluorescence microscopy of SN12C cellsafter incubation with 1613F12 FIG. 27A—Photographs of the mIgG1 isotypecontrol conditions both for the membrane and the intracellular staining.FIG. 27B—Membrane staining. FIG. 27C—Intracellular staining of both Axlreceptor using 1613F12 and of the early endosome marker EEA1. Imageoverlays are presented bellow and co-localizations visualized areindicated by the arrows.

FIGS. 28A and 28B: FIG. 28A represents the cytotoxic activity of thehz1613F12-E-13 on both SN12c (Axl⁺) and MCF-7 (Axl⁻) cell lines. FIG.28B represents the cytotoxic activity of the hz1613F12-G-13 on bothSN12c (Axl⁺) and MCF-7 (Axl⁻) cell lines.

FIGS. 29A and B: In vivo activity of the trastuzumab antibody conjugatedto either E-13 (29A) or G-13 (29B) compounds in the Calu-3 xenograftmodel.

FIGS. 30A to 30C: In vivo activity of the trastuzumab antibodyconjugated to either E-13 (FIG. 30A) or G-13 (FIG. 30B) compounds oralone (FIG. 30C) in the JIMT-1 xenograft model.

EXAMPLES Example 1 Generation of Murine Antibodies Raised Against IGF-1RECD

To generate murine monoclonal antibodies (Mabs) against humanextracellular domain (ECD) of the human IGF-1 receptor (hIGF1R), 5BALB/c mice were immunized 3-times s.c. with 10 μg of the rhIGF-1Rprotein (R&D Systems, Cat No 391-GR). As an alternative, threeadditional immunizations with 10 μg of the murine extracellular domain(ECD) of IGF-1R (R&D Systems, Cat No 6630-GR/Fc) were performed on someanimals. The first immunization was done in presence of Complete FreundAdjuvant (Sigma, St Louis, Md., USA). Incomplete Freund adjuvant (Sigma)was added for following immunizations. Three days prior to the fusion,immunized mice were boosted with 10 μg of the rhIGF-1R protein. Thensplenocytes and lymphocytes were prepared by perfusion of the spleen andby mincing of the proximal lymph nodes, respectively, harvested from 1out of the 5 immunized mice (selected after sera titration of all mice)and fused to SP2/0-Ag14 myeloma cells (ATCC, Rockville, Md., USA). Thefusion protocol is described by Kohler and Milstein (Nature,256:495-497, 1975). Fused cells are then subjected to HAT selection. Ingeneral, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988).Approximately 10 days after the fusion, colonies of hybrid cells werescreened. For the primary screen, supernatants of hybridomas wereevaluated for the secretion of Mabs raised against the IGF-1R ECDprotein by FACS analysis using human breast MCF7 tumor cells (ATCC)and/or monkey COS7 cells (African green monkey kidney-SV40 transformed)which express monkey IGF-1R on their cell surface. More precisely, forthe selection by flow cytometry, 10⁵ cells (either MCF7 or COS7) wereplated in each well of a 96 well-plate in PBS containing 1% BSA and0.01% sodium azide (FACS buffer) at 4° C. After a 2 min centrifugationat 2000 rpm, the buffer was removed and hybridoma supernatants to betested were added. After 20 min of incubation at 4° C., cells werewashed twice and an Alexa 488-conjugated goat anti-mouse antibody 1/500°diluted in FACS buffer (#A11017, Molecular Probes Inc., Eugene, USA) wasadded and incubated for 20 min at 4° C. After a final wash with FACSbuffer, cells were analyzed by FACS (Facscalibur, Becton-Dickinson)after addition of propidium iodide to each tube at a final concentrationof 40 μg/ml. Wells containing cells alone and cells incubated with thesecondary Alexa 488-conjugated antibody were included as negativecontrols. Isotype controls were used in each experiment (Sigma, refM90351MG). At least 5000 cells were assessed to calculate the mean valueof fluorescence intensity (MFI).

Additionally an internalization assay was performed in order to selectonly internalizing antibodies. For this assay, MCF7 tumor cell line wascultured in RMPI 1640 without phenol red with 1% L-glutamine and 10% ofFACS for 3 days before experiment. Cells were then detached usingtrypsin and 100 μl of a cell suspension at 4·10⁵ cell/ml are plated in96-multiwell plates in RPMI1640 without phenol red with 1% L-glutamineand 5% FBS. After a 2 min centrifugation at 2000 rpm, cells wereresuspended in 50 μl of either hybridoma supernatants or controlantibody solutions (positive and isotype controls at 1 μg/ml). After a20 min incubation time at 4° C., cells were centrifuged 2 min at 2000rpm and resuspended in either cold (4° C.) or warm (37° C.) completeculture medium. Cells were then incubated for 2 hours either at 37° C.or at 4° C. Then cells were washed three times with FACS buffer. AnAlexa 488-labeled goat anti-mouse IgG antibody was incubated for 20minutes and cells were washed three times before FACS analysis onpropidium iodide negative cell population.

Following the FACS analysis, two parameters were determined: (i) thedifference of the fluorescent signal detected on the surface of cellsincubated at 4° C. with those obtained with the cells incubated at 37°C. with one hybridoma supernatant and (ii) the percentage of remainingIGF1R on the cell surface.

The percentage of remaining hIGF 1R is calculated as follows: %remaining IGF-1R=(MFI_(Ab 37° C.)/MFI_(Ab 4° C.))×100.

In addition three ELISAs were performed (either before or after cloning)to study the binding of antibodies on the recombinant human (hIGF-1R)and murine (mIGF-1R) proteins, and on the recombinant human InsulinReceptor (hIR) protein. Hybridoma secreting antibody showing binding onrh- and/or rm-IGF-1R and no binding on rhIR were retained. Briefly,96-well ELISA plates (Costar 3690, Corning, N.Y., USA) were coated 100μl/well of either the rhIGF-1R protein (R&D Systems, cat No 391-GR) at0.6 μg/ml or rmIGF-1R protein (R&D Systems, cat No 6630-GR/Fc) at 1μg/ml or rhIR protein (R&D Systems, cat No 1544-IR/CF) at 1 μg/ml in PBSovernight at 4° C. The plates were then blocked with PBS containing 0.5%gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg, Germany) for 2h at 37° C. Once the saturation buffer discarded by flicking plates, 100μl of each supernatant dilution were added to each well (eitherundiluted hybridoma supernatant either supernatant serial dilutions) andincubated for 1 h at 37° C. After three washes, 100 μl horseradishperoxidase-conjugated polyclonal goat anti-mouse IgG (#115-035-164,Jackson Immuno-Research Laboratories, Inc., West Grove, Pa., USA) wasadded at a 1/5000 dilution in PBS containing 0.1% gelatin and 0.05%Tween 20 (w:w) for 1 h at 37° C. Then, ELISA plates were washed 3-timesand the TMB (#UP664782, Uptima, Interchim, France) substrate is added.After a 10 min incubation time at room temperature, the reaction wasstopped using 1 M sulfuric acid and the optical density at 450 nm ismeasured.

Hybridoma secreting antibody of interest were expanded and cloned bylimit dilution. Once isotyped, one clone of each code was expanded andfrozen. Each antibody of interest was produced in in vitro productionsystems named CellLine (Integra Biosciences) for furthercharacterization.

Additional assays to address binding specificity FACS analyses wereperformed on IM9 cells (human IR expressing B lymphoblasts) as well ason hIGF-1R transfected cells versus non transfected cells.

All the data corresponding to the selected antibodies were summarized inTable 9 and demonstrated that the five selected antibodies stronglyrecognize the native human IGF-1R expressed either on MCF-7 breastcancer cells or on transfected cells. They also recognize monkey IGF-1Ron COS-7 cells. These antibodies do not cross react with the humaninsulin receptor highly expressed on IM9 cells. It has to be noticedthat these antibodies poorly recognize the rhIGF-1R ECD protein whendirectly coated to ELISA plates.

TABLE 9 MCF7 Internalisation Assay FACS (SNT at 5 μg/ml) ELISA (SNT at 5μg/ml) (SNT at 5 μg/ml) MFI D.O 450 nm % Δ (MFI IM9 Cos-7 non hybridomarh rm rh MFI remaining 4° C. − MFI (h (monkey Tf Tf cells name IsotypeCNCM IGF-1R IGF-1R Insulin R 4° C. 37° C. rh IGF1R 37° C.) IR⁺) IGF1R⁺)hIGF1R⁺ (h IGF1R⁻) 208F2 IgG1 K I-4757 0.163 0.099 0.140 355 94 27 261 4106 2197 22 212A11 IgG1 K I-4773 0.232 0.102 0.141 390 106 27 284 7 1252187 23 213B10 IgG1 K I-4774 0.399 0.127 0.110 386 115 30 271 7 122 205523 214F8 IgG1 K I-4775 0.349 0.102 0.115 386 111 29 275 7 132 2137 20219D6 IgG1 K I-4736 0.329 0.112 0.106 349 106 30 243 7 114 2110 21

Example 2 Antibody Binding to the Human Native IGF-1R by FACS Analyses

The five murine IGF-1R antibodies were chimerized. The bindingproperties of both the murine and the chimeric IGF-1R antibodies wereevaluated by FACS analyses on the human MCF-7 breast adenocarcinoma cellline (ATCC#HTB-22) using increasing antibody concentrations. For thatpurpose, cells (1×10⁶ cells/ml) were incubated with IGF-1R antibodiesfor 20 min. at 4° C. in FACS buffer (PBS, 0.1% BSA, 0.01% NaN₃). Theywere then washed 3 times and incubated with the appropriate secondaryantibody coupled with Alexa 488 for 20 additional minutes at 4° C. inthe dark before being washed 3 times in FACS buffer. The binding ofanti-IGF-1R antibodies was immediately performed on viable cells whichwere identified using propidium iodide (that stains dead cells). Themaximum of signal intensity obtained with each antibody was designed asB_(max) and expressed in mean of fluorescence intensity (MFI). The EC₅₀of binding expressed in molarity (M) was calculated using a nonlinearregression analysis (GraphPad Prims 4.0).

The titration curve of each murine or chimeric Ab demonstrated that allgenerated antibodies are capable to recognize the native IGF-1R formwith a typical saturation profile (FIG. 1A). In order to rank antibodiesand to compare the binding properties of both murine and chimeric Abs,the binding EC₅₀ of each compound was determined using a non linearregression analysis. The comparison of the EC₅₀ of each murine Ab withits corresponding chimeric form showed that the 2 forms displayed thesame binding properties demonstrating that the Ab chimerization did notaffect IGF-1R recognition (FIG. 1B-C). EC₅₀ and B_(max) values ofchimeric antibodies were summarized in Table 10.

TABLE 10 AC B_(max) EC₅₀ c208F2  981 6.7E−10 c212A11  991 6.7E−10 c214F81069 5.0E−10 c219D6  993 4.7E−10 c213B10 1103 4.4E−10

Example 3 Confirmation of Antibody Specificity by Using Either IGF-1R orIR Transfected Cells or IM9 Cells that Express Significant Levels of IR

In order to confirm the specificity of the generated antibodies forIGF-1R versus IR, stable transfectants expressing either hIGF-1R or hIRwere evaluated by FACS analyses. Briefly, increasing concentrations ofchimeric mAbs were incubated with cells for 20 min at 4° C. in FACSbuffer (PBS, 0.1% BSA, 0.01% NaN₃). Cells were then washed 3 times andincubated with the appropriate secondary antibody coupled with Alexa 488before being incubated for 20 additional minutes at 4° C. in the darkand then washed 3 times in FACS buffer. The binding of anti-IGF-1Rantibodies was immediately performed on viable cells which wereidentified using propidium iodide (that stains dead cells). The bindingEC₅₀ expressed in molarity (M) was calculated using a nonlinearregression analysis (GraphPad Prims 4.0).

Titration curves obtained on the hIGF-1R transfected cell line (FIG. 2A)versus untransfected cells (FIG. 2B) confirmed the binding specificityof chimeric Abs for the human IGF-1R. EC₅₀ and B_(max) values weresummarized in Table 11.

TABLE 11 Ac B_(max) EC₅₀ (M) c208F2 2008 3.2E−10 c212A11 2513 4.4E−10c214F8 2094 2.7E−10 c219D6 2521 5.5E−10 c213B10 2029 3.3E−10

In order to verify the absence of binding of both murine and chimericantibodies on hIR, a stable cell line expressing the human IR (hIR) wasused. The recognition of human cell surface hIR by both murine andchimeric Ab was performed by FACS analyses. Increasing concentration ofeither the murine or the chimeric mAbs were incubated on the hIR⁺transfected cell line for 20 minutes at 4° C. in FACS buffer (PBS, 0.1%BSA, 0.01% NaN₃). Cells were then washed 3 times and incubated with theappropriate secondary antibody coupled with Alexa 488 before beingincubated for 20 additional minutes at 4° C. in the dark and then washed3 times in FACS buffer. The binding of anti-IGF-1R antibodies wasimmediately performed on viable cells which were identified usingpropidium iodide (that stains dead cells). The binding EC₅₀ expressed inmolarity (M) was calculated using a nonlinear regression analysis(GraphPad Prims 4.0). The anti-hIR antibody clone GRO5 was used aspositive controls. The murine and chimeric 9G4 antibodies wereintroduced as irrelevant antibodies.

The high level of expression of hIR on cell surface of the transfectedcells was confirmed using the commercial anti-hIR antibody GRO5 (FIGS.3A and 3B). Even using high concentrations of either the murine (FIG.3A) or the chimeric (FIG. 3B) hIGF-1R Abs, no binding on cell surface ofhIR⁺ transfected cells was observed. These results demonstrated thatneither murine nor chimeric anti-hIGF-1R Abs did recognized the hIR.

This specificity of recognition of hIGF-1R versus IR has also beendemonstrated, by FACS analyses, using IM9 cells, a B-lymphoma cell linethat expresses hIR (FIG. 4). For this FACS analyses, the protocol wasthe same as the one described above and murine antibodies were used inorder to prevent the cross reactivity of the secondary anti-human Ab(IM9 cells express human Ig on their cell surface). Results presented inFIG. 4 demonstrated once again that the expected signal was observedusing the GRO5 anti-hIR antibody while none of the murine antibodyevaluated displayed any significant binding signal on this cell line.

Example 4 Antibody Binding to the Monkey Native IGF-1R by FACS andBiacore Analyses

One of the first pre-requisite for regulatory toxicology studies is tofind a relevant animal specie in order to evaluate the selectedcompound. As the series of antibodies described herein is not able torecognize murine IGF-1R, the most likely specie for toxicologicalevaluation is the non human primate (NHP).

In order to evaluate the binding of anti-IGF-1R antibodies on monkeyIGF-1R, the binding of both murine and chimeric anti-hIGF-1R antibodieswas first evaluated by FACS analyses on COS-7 cell line using increasingantibody concentrations. Cells (1×10⁶ cells/ml) were incubated withanti-IGF-1R antibodies for 20 minutes at 4° C. in FACS buffer (PBS,0.1%, BSA, 0.01% NaN₃). Then, cells were washed 3 times and incubatedwith the appropriate secondary antibody coupled with Alexa 488 beforebeing incubated for 20 additional minutes at 4° C. in the dark andfinally washed 3 times in FACS buffer. The binding of anti-IGF-1Rantibodies was immediately evaluated on viable cells identified usingpropidium iodide (that stains dead cells). The binding EC₅₀ expressed inmolarity (M) was calculated using a nonlinear regression analysis(GraphPad Prims 4.0).

The titration curves obtained on the COS-7 monkey cell line showed that,all the anti-hIGF-1R Abs recognized specifically the IGF-1R expressed onthe surface of the monkey cell line (FIG. 5A). Determination of the,EC₅₀ for each murine and chimeric Abs showed that the 2 forms comparedwell regarding to their binding properties on monkey IGF-1R (FIG. 5B).Those results showed that all the generated anti-hIGF-1R recognized themonkey IGF-1R.

A comparison of binding EC₅₀ on COS-7 cells versus transfected IGF-1Rcells was performed in order to verify the magnitude of chimericantibody recognition on human versus monkey IGF-1R. Results shown inFIG. 5C demonstrated a similar recognition of human and monkey IGF-1Rsby all antibodies.

In order to confirm the recognition on another type of monkey, cellswere transfected with the IGF-1R form Cynomolgus monkey to producesoluble monkey IGF-1R ECD and Biacore experiments were performed withone of the chimeric antibodies (c208F2) in order to compare its bindingproperties either the hIGF-1R or the Cynomolgus IGF-1R.

The recognition experiments were run on a Biacore X100 device using aCM5 sensor chip activated by an anti-Tag His antibody (His capture kitGE Healthcare catalogue number 28-9950-56). More than 11000 RU ofantibodies are chemically grafted on the carboxymethyldextan matrixusing the amine kit chemistry. The experiments were carried out at 25°C. with a flow rate of 30 μl/min using the HBS-EP buffer (GE Healthcare)as the running and sample dilution buffer. The single cycle kineticscheme was used to defined the kinetic parameters of the binding of thechimeric form of the 208F2 antibody (c208F2) on hIGF-1R compared toMacaca IGF-1R

A solution of a soluble recombinant version of the IGF1Rhetero-tetramere composed of 2α chains and the extracellular domains of2β chains expressed with an additional C-terminal 10-His tag, basedeither on the sequence of the human (R&D Systems catalogue number305-GR-50) or of the one of cynomolgus (produced inhouse) was injected 1minute on the second flowcell at a dilution defined to capture around160 RU of antigen. After the capture phase, either the running bufferwas injected 5 times (90 s each injection) or a growing range of 5concentrations of c208F2 were injected (90 s each injection) on bothflowcells. At the end of the fifth injection the running buffer waspassed in order to define the dissociation rate.

The surface was then regenerated with an injection of a 10 mM Glycine,HCl pH 1.5 buffer during 30 s.

The computed signal corresponds to the difference between the responseof the flowcell 2 (with captured IGF-1R) and the response of theflowcell 1 (without any IGF-1R molecules) (FIG. 6).

For each IGF1R molecule (human or cyno), the signal due to theinjections of the growing range of concentrations of c208F2 wascorrected by subtraction of the signal obtained with the 5 injections ofthe buffer (double reference). The resulting sensorgrams were analysedusing the Biaevaluation software with a 1:1 model. The kinetic rates areevaluated either independently (2 kinetics rates of the binding ofc208F2 on each IGF-1R) or commonly (the same kinetic rates of thebinding of c208F2 on the human and the cynomolgus IGF1R). The quality ofthe fitting was assessed by a Chi2/Rmax ratio lower than 0.05 RU.

The kinetics rates of the binding (see Table 12) defined separately foreach IGF-1R are close and a fitting of both sensorgrams with the samekinetic rates is of good quality.

The c208F2 antibody recognizes as well the recombinant human andcynomolgus IGF-1Rs with a dissociation constant (KD) about 0.2 nM. Theaffinities defined in tis study correspond to the functional affinities(avidities) of the antibodies for a level of captured human andcynomolgus IGF-1R around 160 RU.

TABLE 12 IGF1R kon [1/M · s] koff [1/s] Kd [nM] Chi2/Rmax human 1.52E+063.40E−04 0.23 0.045 cynomogus 1.85E+06 3.10E−04 0.17 0.032 Hum. & Cyno.1.52E+06 3.33E−04 0.22 0.039

Example 5 Intrinsic Effect of Generated Antibodies on IGF-1RPhosphorylation

It is well known that antibodies could induce an agonistic effect whenthey bind to tyrosine kinase receptors. As we would not like to selectsuch agonist antibodies, the evaluation of hIGF-1R phosphorylation wasstudied using the chimeric antibodies.

For that purpose, MCF-7 cells were incubated in serum-free mediumovernight. Then, either IGF-1 (100 nM) or Abs to be tested were added(10 μg/ml) for 10 minutes at 37° C. Medium was discarded and cells werescraped in a lysis buffer (pH 7.5) containing 10 mM Tris HCl buffer (pH7.5), 15% NaCl (1 M), 10% detergent mix (10 mM Tris-HCl, 10% Igepallysis buffer) (Sigma Chemical Co.), 5% sodium deoxycholate (SigmaChemical Co.), 1 protease inhibitor cocktail complete TM tablet (Roche),1% phosphatase inhibitor Cocktail Set II (Calbiochem), for 90 min at 4°C. The lysates were clarified by centrifugation at 4° C., heated for 5min at 100° C. and kept at −20° C. or directly loaded on 4-12% SDS-PAGEgels. Incubation of the primary antibody was performed for 2 hr at roomtemperature and then incubation with HRP-linked secondary antibodies wasdone for 1 hr at room temperature. Membranes were washed in TBST priorto visualization of proteins with ECL. Blots were quantified using ImageJ software. Phospho-protein values were normalized with GAPDH.Phosphorylation of hIGF-1R in response to IGF-1 was considered as 100%of stimulation. The effect of anti-hIGF-1R Abs on the phosphorylation ofhIGF-1R was determined as % of phosphorylation induced by IGF-1.

The results described in FIG. 7 represent the mean of the % of pIGF-1Rin response to the chimeric anti-IGF-1R Abs of 3 independent experiments+/−S.D. compared to IGF-1. As illustrated no significant or minor (<10%)phosphorylation of hIGF-1R was detected when MCF-7 cells were incubatedwith 10 μg of anti-IGF-1R Abs.

Example 6 Inhibition of IGF-1R Phosphorylation in Response to IGF-1 byMurine IGF-1R Antibodies

In order to characterize the selected antibodies, their ability toinhibit IGF1-induced phosphorylation was studied. For that purpose,MCF-7 cells were incubated in serum-free medium overnight. Then, cellswere incubated for 5 minutes with murine anti-hIGF-1R Abs beforeaddition of IGF-1 for 2 minutes at 37° C. Medium was discarded and cellswere scraped in a lysis buffer (pH 7.5) containing 10 mM Tris HCl buffer(pH 7.5), 15% NaCl (1 M), 10% detergent mix (10 mM Tris-HCl, 10% Igepallysis buffer) (Sigma Chemical Co.), 5% sodium deoxycholate (SigmaChemical Co.), 1 protease inhibitor cocktail complete TM tablet (Roche),1% phosphatase inhibitor Cocktail Set II (Calbiochem), for 90 min at 4°C. The lysates were clarified by centrifugation at 4° C., heated for 5min at 100° C. and kept at −20° C. or directly loaded on 4-12% SDS-PAGEgels. Incubation of the primary antibody was performed for 2 h at roomtemperature and then incubation with HRP-linked secondary antibodies wasperformed for 1 hr at room temperature. Membranes were washed in TBSTprior to visualization of proteins with ECL. Blots were quantified usingImage J software. Phospho-protein values were normalized with GAPDH.Phosphorylation of hIGF-1R in response to IGF-1 was considered as 100%of stimulation. The effect of anti-hIGF-1R Abs on the phosphorylation ofhIGF-1R was determined as % of phosphorylation induced by IGF-1.

All anti-IGF-1R Abs inhibited strongly hIGF-1R phosphorylation inresponse to IGF-1 (decrease >80%) (FIG. 8). The best inhibitors ofIGF1-induced phosphorylation of hIGF-1R are the m208F2, m212A11 andm214F8 Mabs.

Example 7 Study of IGF-1R Internalization after Binding of the GeneratedIGF-1R Antibodies by FACS Analyses

MCF-7 cells were incubated with 10 μg/ml of chimeric antibodies at 4° C.for 20 min. Then, cells were washed and incubated at 4° C. or 37° C. for4 h. The quantity of cell-surface bound antibody was determined using asecondary antibody. The ΔMFI defined as the difference between MFImeasured at 4° C. and MFI measured at 37° C. after a 4 hour incubationtime corresponded to the quantity of internalized Ab. The ΔMFI waspresented in FIG. 9 and Table 11. The percentage of internalization at10 μg/ml of Ab were calculated as followed 100*(MFI at 4° C.-MFI at 37°C.)/MFI at 4° C. and presented in Table 13.

TABLE 13 Abs % Internalization ΔMFI ΔMFI_ EC₅₀ c208F2 83 288 1.8E−10c212A11 80 322 2.7E−10 c214F8 87 403 2.2E−10 c219D6 80 353 4.4E−10c231B10 85 369 2.3E−10

In order to determine whether antibodies that also recognized the monkeyIGF-1R were able to internalize this receptor, the same internalizationexperiment was performed. Results summarized in Table 14 demonstratedthat all tested antibodies were able to mediate monkey IGF-1Rinternalization.

TABLE 14 Murine Abs Chimeric Abs Abs ΔMFI % internalisation ΔMFI %internalisation 208F2 53 74 52 67 212A11 83 73 98 75 214F8 76 71 98 72219D6 80 71 102 74 213B10 84 74 101 73

The kinetic of cell surface bound antibody decrease was furtherevaluated. For that purpose, MCF-7 cells were seeded in 96-well platesand incubated with 10 μg/ml of murine for 20 min at 4° C. Cells werethen washed to remove unbound antibody and in media at 37° C. for 10,20, 30, 60 or 120 min. At each time point, cells were centrifuged andthen surface labeled on ice with a secondary anti-mouse IgG-Alexa488 todetermine the amount of antibody remaining on the cell surface. Thefluorescence intensity for each murine Ab and for each time point wasnormalized by the signal at 4° C. (% remaining IGF-1R) and fitted to anexponential decay to determine the half life (t1/2). t1/2 was consideredas the time needed to obtain a decrease of 50% of the signal. Asillustrated in FIG. 10, the surface level of all murine Abs droppedrapidly over the first 30 min and the decrease was almost maximum after60 min of incubation (FIG. 10A). The calculated half life was comprisedbetween 10 to 18 min according to the murine Ab (FIG. 10B).

In order to validate that the decrease of the cell surface signal wasdue to Ab internalization and not due to receptor shedding, cells wereincubated with murine Abs for 0, 30 and 60 min a 37° C. (FIG. 11). Cellswere then fixed and permeabilized or not in order to determine cellsurface bound antibody (w/o permeabilization) and total antibody signalcorresponding to cell-surface bound+internalized Ab (withpermeabilization). The quantity of internalized Ab (cytoplasmic) wasdetermined as follow: MFI after permabilization−MFI w/opermeabilization. This experiment showed that the decrease ofcell-surface bound Ab was due to an increase of cytoplasmic Absdemonstrating that Abs were internalized (FIG. 11). In addition, thedegradation of the Abs started after 1 h of incubation as indicated bythe decrease of the signal after permeabilization (Total).

Example 8 Study of IGF-1R Internalization after Binding of the GeneratedIGF-1R Antibodies by Confocal Analyses

To further confirm antibodies internalization, confocal microscopy wasdone to assess the subcellular distribution of antibodies followingcellular trafficking. Cells were incubated with anti-hIGF-1R Abs 37° C.,fixed and permeabilized. Therefore, cells were stained using a secondaryantibody Alexa-488 and with rabbit anti-Lamp-1 antibody that wasrevealed using a secondary anti-Rabbit IgG Alexa 555. Before incubationat 37° C., the murine 208F2 Ab was localized on the membrane of MCF-7cells (FIG. 12A). No colocalization with the lysosome marker, lamp-1 wasnoted using the colocalization highliter plug-in of the Image Jsoftware. The cell surface bound antibody decreased dramatically after15 min of incubation at 37° C. Concomitantly to the decrease of the cellsurface bound antibody, intracellular antibody was detected intovesicles. Rare colocalization with lamp-1 could be observed. After 30min of incubation, the cell surface bound antibody was hardly detected.However, the colocalization of the Ab into lysosome increased. After 1 hof incubation, the intracellular Ab staining decreased as well as thenumber of colocalization with lamp-1. This kinetic of cell surface boundantibody and its intracellular accumulation correlated with the kineticof antibody surface decay measure by FACS. In addition, as alreadydescribed with FACS studies, the degradation of murine Abs started after1 h of incubation by confocal microscopy.

The internalization of all other hIGF-1R murine antibodies and theircolocalization with Lamp-1 was also assessed (FIG. 12B). After 30 min ofincubation at 37° C., intracellular antibody was detected andcolocalization with lamp-1 could be observed indicating that allselected anti-IGF-1R antibodies were effectively internalized intolysosomes.

Example 9 Inhibition of Abs Degradation Using Lysosome Inhibitor,Bafilomycin A1

In order to confirm that antibodies reached the lysosome were they aredegraded, cells were treated or not with bafilomycine A1, a potentinhibitor of lysosome functions. Cells were then incubated with 10 μg/mlof Ab to be tested at 4° C., washed and incubated for 2 h at 37° C. Theinternalized Ab was detected after cell permeabilisation using asecondary anti-mouse IgG-Alexa 488 Ab. Addition of bafilomycine A1prevented the degradation of intracellular Ab (FIG. 13) indicating thatAbs were effectively internalized and degraded into lysosomes.

Example 10 Effect of pH on Antibody-IGF-1R Binding

As antibodies were selected on the bases of their internalizingpotential and shown above to co-localize with early endosomes beforeentering into the lysosomal compartment, an interesting approachconsisted in selecting antibodies for which the stability of theAb/hIGF-1R binding was modulated regarding to pH environment andpreferentially antibodies that dissociated preferentially from IGF-1Rwhen the pH environment became acid. Indeed, the primary differencebetween early endosomes and lysosomes is their luminal pH: in theendosome compartment the pH is approximately 6 while in the lysosomalcompartment the pH is about 4.5.

It is well known that once internalized after ligand binding (IGF1),hIGF-1R returns back to the cell surface through a recycling pathway.

Without being link by a theory, an hypothesis herein described is thatantibodies more prone to be released from their target early at acidicpH will probably favour target recycling to the membrane andconsequently could be considered as better candidates for ADCapproaches. In order to investigate whether some of our antibodiesdisplay such a property and to correlate this property to cytotoxicactivity, the binding of the murine anti-hIGF-1R Mabs on MCF-7 cell linewas done in buffers at different pH. Increasing concentrations of murinemAbs were incubated on MCF-7 cell line for 20 min at 4° C. in differentpH ranging from 5 to 8. Cells were then washed 3 times and incubatedwith the appropriate secondary antibody coupled with Alexa 488 in FACSbuffer. Cells were incubated for 20 additional minutes at 4° C. in thedark and then washed 3 times in FACS buffer. The binding of anti-hIGF-1Rantibodies was immediately performed on viable cells which wereidentified using propidium iodide that stained dead cells. The bindingEC₅₀ expressed in molarity (M) was calculated using a nonlinearregression analysis (GraphPad Prims 4.0). All murine anti-IGF-1Rantibodies selected showed a lower binding capacity at acidic pH asillustrated in FIG. 14.

Example 11 Evaluation of a Humanized Form of the 208F2 Mab

The binding of the first humanized form of the c208F2 mAb was evaluatedon MCF-7, COS-7 and NIH 3T3 IR⁺ cell lines. Increasing concentrations ofm208F2, c208F2 or hz208F2 VH3VL3 were added on each cell line for 20min. at 4° C. Cells were then washed and the binding of the tested mAbwas revealed using the corresponding secondary antibody. In order tovalidate the expression of the human IR on the transfected cell line,the commercial anti-hIR antibody clone GRO5 was used and its recognitionprofile exemplified on (FIG. 15D).

Comparison of the humanized form with either murine or chimeric ones onMCF-7 (FIG. 15A) or monkey COS-7 (FIG. 15B) cells showed close profilesfor the 3 tested forms. The humanisation process did not modify thespecificity of recognition of the antibody that is perfectly comparableto the murine and chimeric forms regarding to the absence of crossreactivity on the human insulin receptor (FIG. 15C).

The calculated EC₅₀ s of the first humanized form of 208F2 on the humancell line MCF-7 and the monkey cell line COS-7 were similar to the onedetermined with either the murine or the chimeric form of the mAb 208F2.

The capacity of the mAb hz208F2 VH3/VL3 to be internalized was assessedby flow cytometry. MCF-7 cells were incubated with 10 μg/ml ofantibodies at 4° C. for 20 min. Then, cells were washed and incubated at4° C. or 37° C. for 4 h. The quantity of cell-surface bound antibody wasdetermined using a secondary antibody. The ΔMFI defined as thedifference between MFI measured at 4° C. and MFI measured at 37° C.after a 4 hour incubation time corresponded to the quantity ofinternalized Ab. The ΔMFI was presented in FIG. 16 and Table 13. Thepercentage of internalization at 10 μg/ml of Ab were calculated asfollowed 100*(MFI at 4° C.-MFI at 37° C.)/MFI at 4° C. and presented inTable 15. Therefore, the humanized hz208F2 VH3/VL3 had similar bindingand internalization properties as the one measured with thecorresponding murine and chimeric 208F2 antibodies.

TABLE 15 ΔMFI % internalization m208F2 294 88 c208F2 278 82 Hz208F2VH3/VL3 344 87

Example 12 Definition of the Dissociation Constant (K_(D)) of theBinding of Five Chimeric Anti-IGF1R Antibodies (c208F2, c213B10,c212A11, c214F8 and c219D6) and a Humanized Version (VH3/VL3) of the208F2 Antibody on a Soluble Recombinant Human IGF1R

The dissociation constants (K_(D)) of the binding of the antibodies on arecombinant soluble human-IGF1R were defined by the ratio between thedissociation rate (k_(off)) and the association rate (k_(on)). Thekinetic experiments were run on a Biacore X100 device using a CM5 sensorchip activated by a mouse anti-Tag His monoclobnal antibody. Around12000 RU of antibodies are chemically grafted on the carboxymethyldextanmatrix using the amine kit chemistry.

The experiments were carried out at 25° C. with a flow rate of 30 μl/minusing the HBS-EP+buffer (GE Healthcare) as the running and sampledilution buffer.

The single cycle kinetic scheme was used to define the kineticparameters of the binding of the anti-IGF1R antibodies on a solublerecombinant human IGF1R captured by its two C-terminal 10 Histidine-tag.

-   -   1—A solution of a soluble recombinant version of the human IGF1R        hetero-tetramere: 2a chains and the extracellular domains of 2β        chains expressed with an additional C-terminal 10-His tag (R&D        Systems catalogue number 305-GR-50) was injected during one        minute on the second flowcell at a concentration of 10 μg/ml. A        mean of 587 RU (with a standard deviation 24 RU) of the soluble        receptor were captured at each of the 24 cycles realised for        this study.    -   2—After the capture phase, either the running buffer was        injected 5 times (90 s each injection) or a growing range of 5        concentrations of one of the six antibodies was injected (90 s        each injection) on both flowcells. At the end of the fifth        injection the running buffer was passed during 5 minutes in        order to define the dissociation rate.    -   3—The surface was then generated with an injection of a 10 mM        Glycine, HCl pH 1.5 buffer during 45 s.

The computed signal corresponds to the difference between the responseof the flowcell 2 (with captured IGF1R) and the response of the flowcell1 (without any IGF1R molecules).

For each IGF1R the signal due to the injections the growing range ofconcentrations of one antibody was corrected by subtraction of thesignal obtained with the 5 injections of the buffer (double reference)see FIG. 17.

The resulting sensorgrams were analysed by the Biaevaluation softwarewith a 1:1 model.

Four experiences were run for each antibody using two different rangesof concentrations: 40, 20, 10, 5 and 2.5 nM for the two firstexperiments and: 24, 12, 6, 3 and 1.5 nM for the two last experimentsrun for each antibody.

For the 6 antibodies tested in this experiment the experimental datafitted well with an 1:1 model with significant k_(off) values when thehigher concentration was defined as a constant and the other fourconcentrations are calculated (see FIG. 18).

The dissociation constants (K_(D)) calculated as the ratio:k_(off)/k_(on) and the half-live of the complexes calculated as theratio: Ln(2)/k_(off) are represented in the FIGS. 19 and 20. Theycorrespond to the mean of the four independent experiments run for eachantibodies. The error bars correspond to the standard errors (n=4) ofthe values.

The dissociation constants are in the range of 10 to 100 pM. The c208F2antibody presents the weaker affinity (higher dissociation constantvalue) for the h-IGF1R (with a K_(D) around 75 pM) and its humanizedversion is at least as good as the chimeric version (with a K_(D) around60 pM). The four other anti-IGF1R chimeric antibodies present a quitesimilar affinity for the hIGF1-R (with a K_(D) around 30 pM). Thedifference of the affinities is principally linked to the dissociationrate or the resultant half life of the complexes. With 208F2 thehalf-life of the complex is between 2 and 3 hour with the chimeric andthe humanized (VH3/VL3) versions. For the four other chimeric antibodiesthe means half lives are between 7.0 and 9.4 h.

These very slow dissociation kinetics are clearly linked to the bivalentstructure of the antibodies which are able to bind simultaneously byboth of their Fab arms to two adjacent h-IGF1R molecules. In this casethe level of captured IGF1R molecules may have an impact on thedissociation rate. The affinities defined in this study correspond tothe functional affinities (or avidities) of the antibodies for a levelof captured h-IGF1R around 600 RU. The 3 fold difference of KD observedbetween data shown above (table 10) and values presented in example 13is linked to a change of the level of capture of hIGF-1R (600 RU versus160 RU in example 4).

Example 13 Generation of 1613F12

To generate murine monoclonal antibodies (Mabs) against humanextracellular domain (ECD) of the Axl receptor, 5 BALB/c mice wereimmunized 5-times s.c. with 15-20·10⁶ CHO-Axl cells and twice with 20 μgof the rh Axl ECD. The first immunization was performed in presence ofComplete Freund Adjuvant (Sigma, St Louis, Md., USA). Incomplete Freundadjuvant (Sigma) was added for following immunizations.

Three days prior to the fusion, immunized mice were boosted with both20·10⁶ CHO-Axl cells and 20 μg of the rhAxl ECD with IFA.

To generate hybridomas, splenocytes and lymphocytes were prepared byperfusion of the spleen and by mincing of the proximal lymph nodes,respectively, harvested from 1 out of the 5 immunized mice (selectedafter sera titration) and fused to SP2/0-Ag14 myeloma cells (ATCC,Rockville, Md., USA). The fusion protocol is described by Kohler andMilstein (Nature, 256:495-497, 1975). Fused cells are then subjected toHAT selection. In general, for the preparation of monoclonal antibodiesor their functional fragments, especially of murine origin, it ispossible to refer to techniques which are described in particular in themanual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988).

Approximately 10 days after the fusion, colonies of hybrid cells werescreened. For the primary screen, supernatants of hybridomas wereevaluated for the secretion of Mabs raised against the Axl ECD proteinusing an ELISA. In parallel, a FACS analysis was performed to selectMabs able to bind to the cellular form of Axl present on the cellsurface using both wt CHO and Axl expressing CHO cells.

As soon as possible, selected hybridomas were cloned by limit dilutionand subsequently screened for their reactivity against the Axl ECDprotein. Cloned Mabs were then isotyped using an Isotyping kit (cat#5300.05, Southern Biotech, Birmingham, Ala., USA). One clone obtainedfrom each hybridoma was selected and expanded.

ELISA assays are performed as followed either using pure hybridomasupernatant or, when IgG content in supernatants was determined,titration was realized starting at 5 μg/ml. Then a ½ serial dilution wasperformed in the following 11 rows. Briefly, 96-well ELISA plates(Costar 3690, Corning, N.Y., USA) were coated 50 μl/well of the rhAxl-Fc protein (R and D Systems, cat No 154-AL) or rhAxl ECD at 2 μg/mlin PBS overnight at 4° C. The plates were then blocked with PBScontaining 0.5% gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg,Germany) for 2 h at 37° C. Once the saturation buffer discarded byflicking plates, 50 μl of pure hybridoma cell supernatants or 50 μl of a5 μg/ml solution were added to the ELISA plates and incubated for 1 h at37° C. After three washes, 50 μl horseradish peroxidase-conjugatedpolyclonal goat anti-mouse IgG (#115-035-164, Jackson Immuno-ResearchLaboratories, Inc., West Grove, Pa., USA) was added at a 1/5000 dilutionin PBS containing 0.1% gelatin and 0.05% Tween 20 (w:w) for 1 h at 37°C. Then, ELISA plates were washed 3-times and the TMB (#UP664782,Uptima, Interchim, France) substrate was added. After a 10 minincubation time at room temperature, the reaction was stopped using 1 Msulfuric acid and the optical density at 450 nm was measured.

For the selection by flow cytometry, 10⁵ cells (CHO wt or CHO-Axl) wereplated in each well of a 96 well-plate in PBS containing 1% BSA and0.01% sodium azide (FACS buffer) at 4° C. After a 2 min centrifugationat 2000 rpm, the buffer was removed and hybridoma supernatants orpurified Mabs (1 μg/ml) to be tested were added. After 20 min ofincubation at 4° C., cells were washed twice and an Alexa 488-conjugatedgoat anti-mouse antibody 1/500° diluted in FACS buffer (#A11017,Molecular Probes Inc., Eugene, USA) was added and incubated for 20 minat 4° C. After a final wash with FACS buffer, cells were analyzed byFACS (Facscalibur, Becton-Dickinson) after addition of propidium iodideto each tube at a final concentration of 40 μg/ml. Wells containingcells alone and cells incubated with the secondary Alexa 488-conjugatedantibody were included as negative controls. Isotype controls were usedin each experiment (Sigma, ref M90351MG). At least 5000 cells wereassessed to calculate the mean value of fluorescence intensity (MFI).

The hybridoma producing the 1613F12 was selected as a candidate.

Example 14 Humanization of 1613F12

The use of mouse antibodies (Mabs) for therapeutic applications inhumans generally results in a major adverse effect, patients raise ahuman anti-mouse antibody (HAMA) response, thereby reducing the efficacyof the treatment and preventing continued administration. One approachto overcome this problem is to humanize mouse Mabs by replacing mousesequences by their human counterpart but without modifying the antigenbinding activity. This can be achieved in two major ways: (i) byconstruction of mouse/human chimeric antibodies where the mouse variableregions are joined to human constant regions (Boulianne et al., 1984)and (ii) by grafting the complementarity determining regions (CDRs) fromthe mouse variable regions into carefully selected human variableregions and then joining these “re-shaped human” variable regions tohuman constant regions (Riechmann et al., 1988).

14.1 Humanization of the Light Chain Variable Domain VL

As a preliminary step, the nucleotide sequence of 1613F12 VL wascompared to the murine germline gene sequences part of the IMGT database(http://www.imgt.org). Murine IGKV16-104*01 and IGKJ5*01 germline geneswere identified. In order to identify the best human candidate for theCDR grafting, the human germline gene displaying the best identity with1613F12 VL murine sequence has been searched. With the help of the IMGTdatabase analyses tools, a possible acceptor human V regions for themurine 1613F12 VL CDRs was identified: IGKV1-27*01 and IGKJ4*02. Inorder to perform the humanization to the light chain variable domaineach residue which is different between the human and mouse sequenceswas given a priority rank order. These priorities (1-4) were used tocreate 11 different humanized variants of the light chain variableregion with up to 14 backmutations.

FR1-IMGT^(®) CDR1-IMGT^(®) FR2-IMGT^(®) CD 1613F12VLDVQITQSPSYLATSPGETITINCRAS KSI......SKY LAWYQEKPGKTNKLLIY SGHomsap IGKV1-27*01 DIQMTQSPSSLSASVGDRVTITCRAS QGI......SNYLAWYQQKPGKVPKLLIY AA  V I     Y AT P ETI  N      E    TN Priority 1 1     3 34 4 433  2      3    33 hz1613F12 (VL1)DIQMTQSPSSLSASVGDRVTITCRAS KSI......SKY LAWYQQKPGKVPKLLIY SGhz1613F12 (VL1I2V) DVQMTQSPSSLSASVGDRVTITCRAS KSI......SKYLAWYQQKPGKVPKLLIY SG hz1613F12 (VL1M4I) DIQITQSPSSLSASVGDRVTITCRASKSI......SKY LAWYQQKPGKVPKLLIY SG hz1613F12 (VL2.1)DVQITQSPSSLSASVGDRVTITCRAS KSI......SKY LAWYQQKPGKVPKLLIY SGhz1613F12 (VL2.1V49T) DVQITQSPSSLSASVGDRVTITCRAS KSI......SKYLAWYQQKPGKTPKLLIY SG hz1613F12 (VL2.1P50N) DVQITQSPSSLSASVGDRVTITCRASKSI......SKY LAWYQQKPGKVNKLLIY SG hz1613F12 (VL2.2)DVQITQSPSSLSASVGDRVTINCRAS KSI......SKY LAWYQQKPGKVPKLLIY SGhz1613F12 (VL2.2V49T) DVQITQSPSSLSASVGDRVTINCRAS KSI......SKYLAWYQQKPGKTPKLLIY SG hz1613F12 (VL2.2P50N) DVQITQSPSSLSASVGDRVTINCRASKSI......SKY LAWYQQKPGKVNKLLIY SG hz1613F12 (VL2.3)DVQITQSPSSLSASVGDRVTINCRAS KSI......SKY LAWYQEKPGKTNKLLIY SGhz1613F12 (VL3) DVQITQSPSYLAASVGDTITINCRAS KSI......SKYLAWYQEKPGKTNKLLIY SG R2-IMGT^(®) FR3-IMGT^(®) 1613F12VL .......STLQSGVP.SRFSGSG..SGTDFTLTISSLEPEDFAMYFC Homsap IGKV1-27*01 .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC                             E   F M F Priority                             4   4 4 2 hz1613F12 (VL1) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL1I2V) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL1M4I) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL2.1) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL2.1V49T) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL2.1P50N) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC hz1613F12 (VL2.2) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYFC hz1613F12 (VL2.2V49T) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYFC hz1613F12 (VL2.2P50N) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYFC hz1613F12 (VL2.3) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYFC hz1613F12 (VL3) .......STLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYFC CDR3-IMGT^(®) FR4-IMGT^(®)1613F12VL QQHHEYPLT FGAGTELELK (SEQ ID NO: 62) Homsap IGKJ4*02        LTFGGGTKVEIK (SEQ ID NO: 108)   A  EL L Priority   3  33 4 hz1613F12 (VL1)QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 70) hz1613F12 (VL1I2V) QQHHEYPLTFGGGTKVEIK (SEQ ID NO: 72) hz1613F12 (VL1M4I) QQHHEYPLT FGGGTKVEIK(SEQ ID NO: 73) hz1613F12 (VL2.1) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 74)hz1613F12 (VL2.1V49T) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 75)hz1613F12 (VL2.1P50N) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 76)hz1613F12 (VL2.2) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 77)hz1613F12 (VL2.2V49T) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 78)hz1613F12 (VL2.2P50N) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 79)hz1613F12 (VL2.3) QQHHEYPLT FGGGTKVEIK (SEQ ID NO: 80) hz1613F12 (VL3)QQHHEYPLT FGAGTELEIK (SEQ ID NO: 81)

14.2 Humanization of the Heavy Chain Variable Domain VH

In order to identify the best human candidate for the CDR grafting, themouse and human germline genes displaying the best identity with 1613F12VH were searched. The nucleotide sequence of 1613F12 VH was aligned withboth mouse and human germline gene sequences by using the sequencealignment software “IMGT/V-QUEST” which is part of the IMGT database.Alignments of amino acid sequences were also performed to verify theresults of the nucleotide sequence alignment using the “Align X”software of the VectorNTI package. The alignment with mouse germlinegenes showed that the mouse germline V-gene IGHV14-3*02 and J-geneIGHJ2*01 are the most homologue mouse germline genes. Using the IMGTdatabase the mouse D-gene germline IGHD1-1*01 was identified ashomologous sequence. In order to select an appropriate human germlinefor the CDR grafting, the human germline gene with the highest homologyto 1613F12 VH murine sequence was identified. With the help of IMGTdatabases and tools, the human IGHV1-2*02 germline gene and humanIGHJ5*01 J germline gene were selected as human acceptor sequences forthe murine 1613F12 VH CDRs. In order to perform the humanization to theheavy chain variable domain each residue which is different between thehuman and mouse sequences was given a priority rank order (1-4). Thesepriorities were used to create 20 different humanized variants of theheavy chain variable region with up to 18 backmutations,

FR1-IMGT^(®) CDR1-IMGT^(®) FR2-IMGT^(®) CD (1-26) (27-38) (39-55) (1613F12 EVHLQQSGA.ELVKPGASVKLSCTAS GFNI....RDTY IHWVKQRPEQGLEWIGR LDHomsap IGHV1-2*02 QVQLVQSGA.EVKKPGASVKVSCKAS GYTF....TGYYMHWVRQAPGQGLEWMGW IN E H Q      LV       L  T I   K R E     I R Priority3 2 3      33       3  3 1   3 4 4     3 2 hz1613F12 (VH1)QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTY MHWVRQAPGQGLEWMGW LDhz1613F12 (VH1M39I) QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTYIHWVRQAPGQGLEWMGW LD hz1613F12 (VH1W55RN66K) QVQLVQSGA.EVKKPGASVKVSCKASGFNI....RDTY MHWVRQAPGQGLEWMGR LD hz1613F12 (VH1I84S)QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTY MHWVRQAPGQGLEWMGW LDhz1613F12 (VH1S85N) QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTYMHWVRQAPGQGLEWMGW LD hz1613F12 (VH1I84NS85N) QVQLVQSGA.EVKKPGASVKVSCKASGFNI....RDTY MHWVRQAPGQGLEWMGW LD hz1613F12 (VH2.1)QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTY IHWVRQAPGQGLEWMGW LDhz1613F12 (VH2.1Q3H) QVHLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTYIHWVRQAPGQGLEWMGW LD hz1613F12 (VH2.1W55R) QVQLVQSGA.EVKKPGASVKVSCKASGFNI....RDTY IHWVRQAPGQGLEWMGR LD hz1613F12 (VH2.1N66K)QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTY IHWVRQAPGQGLEWMGW LDhz1613F12 (VH2.1W55RN66K) QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTYIHWVRQAPGQGLEWMGR LD hz1613F12 (VH2.1R80S) QVQLVQSGA.EVKKPGASVKVSCKASGFNI....RDTY IHWVRQAPGQGLEWMGW LD hz1613F12 (VH2.1N66KR80S)QVQLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTY IHWVRQAPGQGLEWMGW LDhz1613F12 (VH2.2) QVHLVQSGA.EVKKPGASVKVSCKAS GFNI....RDTYIHWVRQAPGQGLEWMGW LD hz1613F12 (VH2.2M89L) QVHLVQSGA.EVKKPGASVKVSCKASGFNI....RDTY IHWVRQAPGQGLEWMGW LD hz1613F12 (VH2.3)QVQLQQSGA.EVKKPGASVKLSCTAS GFNI....RDTY IHWVRQAPGQGLEWMGW LDhz1613F12 (VH2.3W55R) QVQLQQSGA.EVKKPGASVKLSCTAS GFNI....RDTYIHWVRQAPGQGLEWMGR LD hz1613F12 (VH2.3Q3HW55R) QVHLQQSGA.EVKKPGASVKLSCTASGFNI....RDTY IHWVRQAPGQGLEWMGR LD hz1613F12 (VH2.4)QVQLQQSGA.EVKKPGASVKLSCTAS GFNI....RDTY IHWVRQAPGQGLEWIGR LDhz1613F12 (VH3) EVHLQQSGA.ELVKPGASVKLSCTAS GFNI....RDTYIHWVKQAPGQGLEWIGR LD CDR2-IMGT^(®) FR3-IMGT^(®) (56-65) (66-104) 1613F12LDPA..NGHT KYGPNFQ.GRATMTSDTSSNTAYLQLSSLTSEDTAVYYC Homsap IGHV1-2*02INPN..SGGT NYAQKFQ.GRVTMTRDTSISTAYMELSRLRSDDTAVYYCK GPN     A   S   SN   LQ  S T E Prority2 344     4   2   11   33  4 4 4 hz1613F12 (VH1) LDPA..NGHTNYAQKFQ.GRVTMTRDTSISTAYMELSRLRSDDTAVYYC hz1613F12 (VH1M39I) LDPA..NGHTNYAQKFQ.GRVTMTRDTSISTAYMELSRLRSDDTAVYYC hz1613F12 (VH1W55RN66K)LDPA..NGHT KYAQKFQ.GRVTMTRDTSISTAYMELSRLRSDDTAVYYC hz1613F12 (VH1I84S)LDPA..NGHT NYAQKFQ.GRVTMTRDTSSSTAYMELSRLRSDDTAVYYC hz1613F12 (VH1S85N)LDPA..NGHT NYAQKFQ.GRVTMTRDTSINTAYMELSRLRSDDTAVYYChz1613F12 (VH1I84NS85N) LDPA..NGHTNYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1) LDPA..NGHTNYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1Q3H) LDPA..NGHTNYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1W55R) LDPA..NGHTNYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1N66K) LDPA..NGHTKYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1W55RN66K)LDPA..NGHT KYAQKFQ.GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.1R80S)LDPA..NGHT NYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYChz1613F12 (VH2.1N66KR80S) LDPA..NGHTKYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.2) LDPA..NGHTKYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.2M89L) LDPA..NGHTKYAQKFQ.GRVTMTSDTSSNTAYLELSRLRSDDTAVYYC hz1613F12 (VH2.3) LDPA..NGHTKYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.3W55R) LDPA..NGHTKYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.3Q3HW55R)LDPA..NGHT KYAQKFQ.GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC hz1613F12 (VH2.4)LDPA..NGHT KYAQKFQ.GRVTMTSDTSSNTAYLELSRLRSDDTAVYYC hz1613F12 (VH3)LDPA..NGHT KYGQKFQ.GRVIMISDISSNTAYLQLSRLRSDDTAVYYC CDR3-IMGT^(®)FR4-IMGT^(®) 1613F12VH ARGAYYYGSSGLFYFDY WGQGTTLSVSS (SEQ ID NO: 63)Homsap IGHJ5*01 WGQGTLVTVSS (SEQ ID NO: 109)      TLS Prority      444hz1613F12 (VH1) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 82)hz1613F12 (VH1M39I) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 84)hz1613F12 (VH1W55RN66K) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 85)hz1613F12 (VH1I84S) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 86)hz1613F12 (VH1S85N) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 87)hz1613F12 (VH1I84NS85N) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 88)hz1613F12 (VH2.1) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 89)hz1613F12 (VH2.1Q3H) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 90)hz1613F12 (VH2.1W55R) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 91)hz1613F12 (VH2.1N66K) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 92)hz1613F12 (VH2.1W55RN66K) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 93)hz1613F12 (VH2.1R80S) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 94)hz1613F12 (VH2.1N66KR80S) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 95)hz1613F12 (VH2.2) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 96)hz1613F12 (VH2.2M89L) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 97)hz1613F12 (VH2.3) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 98)hz1613F12 (VH2.3W55R) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 99)hz1613F12 (VH2.3Q3HW55R) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 100)hz1613F12 (VH2.4) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 101)hz1613F12 (VH3) ARGAYYYGSSGLFYFDY WGQGTLVTVSS (SEQ ID NO: 102)

Example 15 Axl Binding Specificity

In this example, the binding of 1613F12 was first studied on therhAxl-Fc protein. Then, its binding on the two other members of the TAMfamily, rhDtk-Fc and rhMer-Fc, was studied.

Briefly, the recombinant human Axl-Fc (R and D systems, cat No154AL/CF), rhDtk (R and D Systems, cat No 859-DK) or rhMer-Fc (R and DSystems, cat No 891-MR) proteins were coated overnight at 4° C. toImmulon II 96-well plates and, after a 1 h blocking step with a 0.5%gelatine solution, 1613F12 was added for an additional 1 h at 37° C. atstarting concentration of 5 μg/ml (3.33 10⁻⁸ M). Then ½ serial dilutionswere done over 12 columns. Plates were washed and a goat anti-mouse(Jackson) specific IgG-HRP was added for 1 h at 37° C. Reactiondevelopment was performed using the TMB substrate solution. The isotypecontrol antibody mIgG1 and the commercial anti-Axl Mab 154 antibody werealso used in parallel. Coating controls were performed in presence of agoat anti-human IgG Fc polyclonal serum labelled with HRP (Jackson, ref109-035-098) and/or in presence of a HRP-coupled anti-Histidine antibody(R and D Systems, ref: MAB050H).

Results are represented in FIGS. 24A, 24B and 24C, respectively.

This example shows that 1613F12 only binds to the rhAxl-Fc protein anddoes not bind on the two other members of the TAM family, rhDtk orrhMer. No cross-specificity of binding of 1613F12 is observed betweenTAM members. No non specific binding was observed in absence of primaryantibody (diluent). No binding was observed in presence of the isotypecontrol antibody.

Example 16 1613F12 Recognized the Cellular Form of Axl Expressed onHuman Tumor Cells

Cell surface Axl expression level on human tumor cells was firstestablished using a commercial Axl antibody (R and D Systems, ref:MAB154) in parallel of calibration beads to allow the quantification ofAxl expression level. Secondly, binding of the cell-surface Axl wasstudied using 1613F12.

For cell surface binding studies, two fold serial dilutions of a 10μg/ml (6.66 10⁻⁸M) primary antibody solution (1613F12, MAB154 or mIgG1isotype control 9G4 Mab) are prepared and are applied on 2·10⁵ cells for20 min at 4° C. After 3 washes in phosphate-buffered saline (PBS)supplemented with 1% BSA and 0.01% NaN₃, cells were incubated withsecondary antibody Goat anti-mouse Alexa 488 ( 1/500° dilution) for 20minutes at 4° C. After 3 additional washes in PBS supplemented with 1%BSA and 0.1% NaN₃, cells were analyzed by FACS (Facscalibur,Becton-Dickinson). At least 5000 cells were assessed to calculate themean value of fluorescence intensity.

For quantitative ABC determination using MAB154, QIFIKIT® calibrationbeads are used. Then, the cells are incubated, in parallel with theQIFIKIT® beads, with Polyclonal Goat Anti-Mouse Immunoglobulins/FITC,Goat F(ab′)₂, at saturating concentration. The number of antigenic siteson the specimen cells is then determined by interpolation of thecalibration curve (the fluorescence intensity of the individual beadpopulations against the number of Mab molecules on the beads.

16.1. Quantification of Cell-Surface Axl Expression Level

Axl expression level on the surface of human tumor cells was determinedby flow cytometry using indirect immunofluorescence assay (QIFIKIT®method (Dako, Denmark), a quantitative flow cytometry kit for assessingcell surface antigens. A comparison of the mean fluorescence intensity(MFI) of the known antigen levels of the beads via a calibration graphpermits determination of the antibody binding capacity (ABC) of the celllines.

Table 16 presents Axl expression level detected on the surface ofvarious human tumor cell lines (SN12C, Calu-1, MDA-MB435S, MDA-MB231,NCI-H125, MCF7, Panc1) as determined using QIFIKIT® using the MAB154 (Rand D Systems). Values are given as Antigen binding complex (ABC).

TABLE 16 MCF7 NCI-H125 MDA-MB-435S Panc1 MDA-MB-231 Calu-1 SN12C TumorBreast NSCLC Breast Pancreas Breast Lung Renal type/organ ABC 71 5 54017 814 36 809 61 186 >100 000 >100 000 (Qifikit)

Results obtained with MAB154 showed that Axl receptor is expressed atvarious levels depending of the considered human tumor cell.

16.2. Axl Detection by 1613F12 on Human Tumor Cells

More specifically, Axl binding was studied using 1613F12.

1613F12 dose response curves were prepared. MFIs obtained using thevarious human tumor cells were then analysed with Prism software. Dataare presented in FIG. 25.

Data indicate that 1613F12 binds specifically to the membrane Axlreceptor as attested by the saturation curve profiles. However differentintensities of labelling were observed, revealing variable levels ofcell-surface Axl receptor on human tumor cells. No binding of Axlreceptor was observed using MCF7 human breast tumor cell line.

Example 17 Validation of hz1613F12 vs. m1613F12

In order to establish whether hz1613F12 was comparable to its murineform, binding experiments were performed by ELISA using rhAxl-Fc proteinassays.

In this assay, 96 well plates (Immulon II, Thermo Fisher) were coatedwith a 5 μg/ml of 1613F12 solution in 1×PBS, overnight at 4° C. After asaturation step, a range of rh Axl-Fc protein (R and D Systems, ref:154-AL) is incubated for 1 hour at 37° C. on the coated plates. For therevelation step, a biotinylated-Axl antibody (in house product) wasadded at 0.85 μg/ml for 1 hour at 37° C. This Axl antibody belongs to adistinct epitopic group. Then an avidin-horseradish peroxidase solutionat 1/20000 in diluent buffer is added to the wells. Then the TMBsubstrate solution is added for 5 min. After addition of the peroxydasestop solution, the absorbance at 405 nm was measured with a microplatereader.

FIG. 26 shows that both murine and humanized versions of 1613F12 bindsimilarly the rhAxl-Fc protein.

Example 18 1613F12 Internalization Study Using FluorescentImmunocytochemistry Labelling

Complementary internalization results are obtained by confocalmicroscopy using indirect fluorescent labelling method.

Briefly, SN12C tumor cell line was cultured in RMPI1640 with 1%L-glutamine and 10% of FCS for 3 days before experiment. Cells were thendetached using trypsin and plated in 6-multiwell plate containingcoverslide in RPMI1640 with 1% L-glutamine and 5% FCS. The next day,1613F12 was added at 10 μg/ml. Cells treated with an irrelevant antibodywere also included. The cells were then incubated for 1 h and 2 h at 37°C., 5% CO₂. For T 0 h, cells were incubated for 30 minutes at 4° C. todetermine antibody binding on cell surface. Cells were washed with PBSand fixed with paraformaldehyde for 15 minutes. Cells were rinsed andincubated with a goat anti-mouse IgG Alexa 488 antibody for 60 minutesat 4° C. to identify remaining antibody on the cell surface. To followantibody penetration into the cells, cells were fixed and permeabilizedwith saponin. A goat anti-mouse IgG Alexa 488 (Invitrogen) was used tostained both the membrane and the intracellular antibody. Earlyendosomes were identified using a rabbit polyclonal antibody againstEEA1 revealed with a goat anti-rabbit IgG-Alexa 555 antibody(Invitrogen). Cells were washed three times and nuclei were stainedusing Draq5. After staining, cells were mounted in Prolong Gold mountingmedium (Invitrogen) and analyzed by using a Zeiss LSM 510 confocalmicroscope.

Photographs are presented in FIGS. 27A, 27B, and 27C.

Images were obtained by confocal microscopy. In presence of the mIgG1isotype control (9G4), neither membrane staining nor intracellularlabelling is observed (FIG. 27A). A progressive loss of the membraneanti-Axl labelling is observed as soon as after 1 h incubation of theSN12C cells with 1613F12 (FIG. 27B). Intracellular accumulation of1613F12 antibody is clearly observed at 1 h and 2 h (FIG. 27C).Intracellular antibody co-localizes with EEA1, an early endosome marker.These photographs confirm the internalization of 1613F12 into SN12Ccells.

Example 19 Synthesis of the Drugs of the Invention

The following abbreviations are used in the following examples:

-   aq. aqueous-   ee enantiomeric excess-   equiv equivalent-   ESI Electrospray ionisation-   LC/MS Liquid Chromatography coupled with Mass Spectrometry-   HPLC High Performance Liquid Chromatography-   NMR Nuclear Magnetic Resonance-   sat. saturated-   UV ultraviolet

Compound 1(S)-2-((S)-2-((3-aminopropyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,bis trifluoroacetic acid

Compound 1A: (4R,5S)-4-methyl-5-phenyl-3-propanoyl-1,3-oxazolidin-2-one

(4R,5S)-4-methyl-5-phenyl-1,3-oxazolidin-2-one (5.8 g, 32.7 mmol, 1.00equiv) was dissolved in tetrahydrofuran (THF, 120 mL) in an inertatmosphere. The mixture was cooled to −78° C. and n-butyllithium (14.4mL) was added drop-wise. After agitation for 30 minutes at −78° C.,propanoyl chloride (5.7 mL) was added. Agitation was continued for 30minutes at −78° C. then overnight at ambient temperature. The reactionmixture was concentrated then re-dissolved in 200 mL of water. The pH ofthe solution was adjusted to 7 with sodium bicarbonate saturated aqueoussolution. This aqueous phase was extracted 3 times with 100 mL of ethylacetate (EtOAc). The organic phases were combined, dried over sodiumsulfate, filtered and concentrated to yield 6.8 g (89%) of compound 1Ain the form of a yellow oil.

Compound 1B: tert-butyl(2S)-2-[(1R,2R)-1-hydroxy-2-methyl-3-[(4R,5S)-4-methyl-2-oxo-5-phenyl-1,3-oxazolidin-3-yl]-3-oxopropyl]pyrrolidine-1-carboxylate

Compound 1A (17.6 g, 75.45 mmol, 1.00 equiv) was dissolved indichloromethane (DCM, 286 mL) in an inert atmosphere. This solution wascooled with an ice bath. Triethylamine (TEA, 12.1 mL, 1.15 equiv) andBu₂BOTf (78.3 mL, 1.04 equiv) were added drop-wise whilst holding thetemperature of the reaction mixture below 2° C. Agitation was continuedat 0° C. for 45 minutes, after which the reaction was cooled to −78° C.A solution of tert-butyl (2S)-2-formylpyrrolidine-1-carboxylate (8.5 g,42.66 mmol, 0.57 equiv) in DCM (42 mL) was added drop-wise. Agitationwas continued for 2 hours at −78° C., then for 1 hour at 0° C. andfinally 1 hour at ambient temperature. The reaction was neutralised with72 mL of phosphate buffer (pH=7.2-7.4) and 214 mL methanol, and cooledto 0° C. A solution of 30% hydrogen peroxide in methanol (257 mL) wasadded drop-wise whilst maintaining the temperature below 10° C.Agitation was continued for 1 hour at 0° C. The reaction was neutralisedwith 142 mL of water, then concentrated under reduced pressure. Theresulting aqueous solution was extracted 3 times with 200 mL EtOAc. Theorganic phases were combined, dried over sodium sulfate, filtered andconcentrated. The residue was purified on a silica column with a mixtureof EtOAc and petroleum ether (EtOAc:PE=1:8) to yield 13.16 g (40%) ofcompound 1B in the form of a colourless oil.

Compound 1C:(2R,3R)-3-[(2S)-1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl]-3-hydroxy-2-methylpropanoicacid

Compound 1B (13.16 g, 30.43 mmol, 1.00 equiv) was dissolved in THF (460mL) in the presence of hydrogen peroxide (30% in water, 15.7 mL), thencooled with an ice bath. An aqueous solution of lithium hydroxide (0.4mol/L, 152.1 mL) was added drop-wise whilst holding the reactiontemperature below 4° C. The reaction mixture was agitated 2.5 hours at0° C. An aqueous solution of Na₂SO₃ (1 mol/L, 167.3 mL) was addeddrop-wise whist holding the temperature at 0° C. The reaction mixturewas agitated 14 hours at ambient temperature, then neutralised with 150mL of cold sodium bicarbonate saturated solution and washed 3 times with50 mL of DCM. The pH of the aqueous solution was adjusted to 2-3 with a1M aqueous solution of KHSO₄. This aqueous solution was extracted 3times with 100 mL of EtOAc. The organic phases were combined, washedonce with saturated NaCl solution, dried over sodium sulfate, filteredand concentrated to yield 7.31 g (88%) of compound 1C in the form of acolourless oil.

Compound 1D:(2R,3R)-3-[(2S)-1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl]-3-methoxy-2-methylpropanoicacid

Compound 1C (7.31 g, 26.74 mmol, 1.00 equiv) was dissolved in an inertatmosphere in THF (135 mL) in the presence of iodomethane (25.3 mL). Thereaction medium was cooled with an ice bath after which NaH (60% in oil,4.28 g) was added in portions. The reaction was left under agitation 3days at 0° C. and then neutralised with 100 mL of sodium bicarbonatesaturated aqueous solution and washed 3 times with 50 mL ether. The pHof the aqueous solution was adjusted to 3 with 1M aqueous KHSO₄solution. This aqueous solution was extracted 3 times with 100 mL ofEtOAc. The organic phases were combined, washed once with 100 mL ofNa₂S₂O₃ (5% in water), once with NaCl-saturated solution, then driedover sodium sulfate, filtered and concentrated to yield 5.5 g (72%) ofcompound 1D in the form of a colourless oil.

Compound 1E: N-methoxy-N-methyl-2-phenylacetamide

2-phenylacetic acid (16.2 g, 118.99 mmol, 1.00 equiv) was dissolved indimethylformamide (DMF, 130 mL) then cooled to −10° C. Diethylphosphorocyanidate (DEPC, 19.2 mL), methoxy(methyl)amine hydrochloride(12.92 g, 133.20 mmol, 1.12 equiv) and triethylamine (33.6 mL) wereadded. The reaction mixture was agitated 30 minutes at −10° C. then 2.5hours at ambient temperature. It was then extracted twice with 1 litreof EtOAc. The organic phases were combined, washed twice with 500 mL ofNaHCO₃ (sat.), once with 400 mL of water, then dried over sodiumsulfate, filtered and concentrated. The residue was purified on a silicacolumn with an EtOAc and PE mixture (1:100 to 1:3) to yield 20.2 g (95%)of compound 1E in the form of a yellow oil.

Compound 1F: 2-phenyl-1-(1,3-thiazol-2-yl)ethan-1-one

Tetramethylethylenediamine (TMEDA, 27.2 mL) was dissolved in THF 300 mL)in an inert atmosphere, then cooled to −78° C. before the drop-wiseaddition of n-BuLi (67.6 mL, 2.5 M). 2-bromo-1,3-thiazole (15.2 mL) wasadded drop-wise and agitation was continued 30 minutes at −78° C.Compound 1E (25 g, 139.50 mmol, 1.00 equiv) dissolved in THF (100 mL)was added drop-wise. Agitation was continued for 30 minutes at −78° C.then 2 hours at −10° C. The reaction was neutralised with 500 mL ofKHSO₄ (sat.), then extracted 3 times with 1 litre of EtOAc. The organicphases were combined, washed twice with 400 mL water and twice with 700mL of NaCl (sat.), then dried over sodium sulfate, filtered andconcentrated. The residue was purified on a silica column with a mixtureof EtOAc and PE (1:100 to 1:10) to yield 25 g (88%) of compound 1F inthe form of a yellow oil.

Compound 1G: (1R)-2-phenyl-1-(1,3-thiazol-2-yl)ethan-1-ol

In an inert atmosphere, a solution of compound 1F (15 g, 73.8 mmol, 1.00equiv.) in ether (300 mL) was added drop-wise to(+)-B-chlorodiisopinocampheylborane ((+)-Ipc₂BCl, 110.8 mL). Thereaction mixture was agitated 24 hours at 0° C., then neutralised with300 mL of a (1:1) mixture of NaOH (10% in water) and H₂O₂(30% in water),and finally extracted three times with 500 mL of EtOAc. The organicphases were combined, washed twice with 300 mL of K₂CO₃ (sat.) and oncewith 500 mL of NaCl (sat.), then dried over sodium sulfate, filtered andconcentrated. The residue was purified on a silica column with a mixtureof EtOAc and PE (1:20 to 1:2) to yield 6.3 g (42%) of compound 1G in theform of a white solid.

Compound 1H: 2-[(1S)-1-azido-2-phenylethyl]-1,3-thiazole

Compound 1G (6 g, 29.23 mmol, 1.00 equiv.) was dissolved in an inertatmosphere in THF (150 mL) in the presence of triphenylphosphine (13 g,49.56 mmol, 1.70 equiv.), then cooled to 0° C. Diethylazodicarboxylate(DEAD, 7.6 mL) was added drop-wise, followed by diphenylphosphorylazide(DPPA, 11 mL), the cold bath was then removed and the solution was leftunder agitation 48 hours at ambient temperature. The medium wasconcentrated under reduced pressure. The residue was purified on asilica column with a mixture of EtOAc and PE (1:100 to 1:30) to yield 8g of partly purified compound 1H in the form of a yellow oil. Compound1H was used as such in the following step.

Compound 1I: tert-butylN-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamate

Compound 1H (6.5 g, 28.2 mmol, 1.00 equiv) was dissolved in an inertatmosphere in THF (100 mL) in the presence of triphenylphosphine (6.5 g,33.9 mmol, 1.20 equiv.), and heated to 50° C. for 2 hours. Ammonia (70mL) was then added and heating was continued for 3 hours. The reactionwas cooled, neutralised with 500 mL water, then extracted 3 times with500 mL of EtOAc. The organic phases were combined and extracted twicewith 500 mL of 1N HCl. The aqueous phases were combined, brought to pH8-9 by adding a sodium hydroxide solution (10% in water), then extracted3 times with 500 mL of DCM. The organic phases were combined, dried oversodium sulfate, filtered and concentrated to yield 4.8 g (83%) of(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethan-1-amine in the form of a yellowoil. This compound was then protected with a Boc group((tert-butoxy)carbonyl) so that it could be purified. It was dissolvedin an inert atmosphere in 1,4-dioxane (40 mL), then cooled to 0° C.(Boc)₂O (10.26 g, 47.01 mmol, 2.00 equiv) diluted in 20 mL of1,4-dioxane was added drop-wise. The cold bath was removed and thesolution left under agitation overnight at ambient temperature beforebeing neutralised with 300 mL of water and extracted twice with 500 mLof EtOAc. The organic phases were combined, dried over sodium sulfate,filtered and concentrated. The residue was purified on a silica columnwith a mixture of EtOAc and PE (1:100 to 1:20, ee=93%). It was thenrecrystallized in a hexane/acetone mixture (˜5-10/1, 1 g/10 mL) to yield6 g (84%) of compound 1I in the form of a white solid (ee >99%).

Compound 1J: tert-butyl(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamoyl]ethyl]pyrrolidine-1-carboxylate

Compound 1I (3 g, 9.86 mmol, 1.00 equiv) was dissolved in an inertatmosphere in 10 mL DCM. Trifluoroacetic acid (TFA, 10 mL) was added andthe solution left under agitation overnight at ambient temperature, thenconcentrated under reduced pressure to yield 2.0 g (64%) of(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethan-1-amine; trifluoroacetic acid inthe form of a yellow oil. This intermediate was re-dissolved in 20 mL ofDCM after which compound 1D (1.8 g, 6.26 mmol, 1.05 equiv), DEPC (1.1 g,6.75 mmol, 1.13 equiv) and diisopropylethylamine (DIEA, 1.64 g, 12.71mmol, 2.13 equiv) were added. The reaction mixture was left underagitation overnight at ambient temperature, then concentrated underreduced pressure. The residue was purified on a silica column with amixture of EtOAc and PE (1:100 to 1:3) to yield 2.3 g (81%) of compound1J in the form of a pale yellow solid.

Compound 1K:(2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanamide;trifluoroacetic acid

Compound 1J (2.25 g, 4.75 mmol, 1.00 equiv) was dissolved in an inertatmosphere in 10 mL of DCM. TFA (10 mL) was added and the solution leftunder agitation overnight at ambient temperature, then concentratedunder reduced pressure to yield 2.18 g (94%) of compound 1K in the formof a yellow oil.

Compound 1L: (2S,3S)-2-(benzylamino)-3-methylpentanoic acid

(2S,3S)-2-amino-3-methylpentanoic acid (98.4 g, 750 mmol, 1.00 equiv)was added at ambient temperature and in portions to a 2N sodiumhydroxide solution (375 mL). Benzaldehyde (79.7 g, 751.02 mmol, 1.00equiv) was quickly added and the resulting solution was agitated 30minutes. Sodium borohydride (10.9 g, 288.17 mmol, 0.38 equiv) was addedin small portions, whilst holding the temperature at between 5 and 15°C. Agitation was continued for 4 hours at ambient temperature. Thereaction mixture was diluted with 200 mL of water, then washed twicewith 200 mL of EtOAc. The pH of the aqueous solution was adjusted to 7with a 2N hydrochloric acid solution. The formed precipitate wascollected by filtering and gave 149.2 g (90%) of compound 1L in the formof a white solid.

Compound 1M: (2S,3S)-2-[benzyl(methyl)amino]-3-methylpentanoic acid

Compound 1L (25 g, 112.97 mmol, 1.00 equiv) was dissolved in an inertatmosphere in formic acid (31.2 g) in the presence of formaldehyde(36.5% in water, 22.3 g). The solution was agitated 3 hours at 90° C.then concentrated under reduced pressure. The residue was triturated in250 mL of acetone, then concentrated. This trituration/evaporationoperation was repeated twice with 500 mL of acetone to yield 21.6 g(81%) of compound 1M in the form of a white solid.

Compound 1N: (2S,3S)-2-[benzyl(methyl)amino]-3-methylpentan-1-ol

LiAlH₄ (0.36 g) was suspended in 10 mL of THF in an inert atmosphere at0° C. Compound 1M (1.5 g, 6.37 mmol, 1.00 equiv) was added in smallportions whilst holding the temperature at between 0 and 10° C. Thereaction mixture was agitated 2 hours at 65° C., then again cooled to 0°C. before being neutralised with successive additions of 360 μL ofwater, 1 mL of 15% sodium hydroxide and 360 μL of water. The aluminiumsalts which precipitated were removed by filtering. The filtrate wasdried over sodium sulfate, filtered and concentrated. The residue waspurified on a silica column with a mixture of EtOAc and PE (1:50) toyield 820 mg (58%) of compound 1N in the form of a pale yellow oil.

Compound 1O: (2S,3S)-2-[benzyl(methyl)amino]-3-methylpentanal

Oxalyl chloride (0.4 mL) was dissolved in DCM (15 mL) in an inertatmosphere. The solution was cooled to −70° C. and a solution ofdimethylsulfoxide (DMSO (0.5 mL) in DCM (10 mL) was added drop-wise for15 minutes. The reaction mixture was agitated 30 minutes after which asolution of compound 1N (820 mg, 3.70 mmol, 1.00 equiv) in DCM (10 mL)was added drop-wise for 15 minutes. The reaction mixture was agitated afurther 30 minutes at low temperature, then triethylamine (2.5 mL) wasslowly added. The reaction mixture was agitated 1 hour at −50° C., thecold bath was then removed and the reaction neutralised with 25 mL ofwater whilst allowing the temperature to return to normal. The solutionwas washed once with 30 mL of NaCl-saturated aqueous solution, thendried over sodium sulfate, filtered and concentrated. The residue waspurified on a silica column with a mixture of EtOAc and PE (1:200) toyield 0.42 g (52%) of compound 1O in the form of a yellow oil.

Compound 1P: (2S,3S)—N-benzyl-1,1-dimethoxy-N,3-dimethylpentan-2-amine

Compound 10 (4.7 g, 21.43 mmol, 1.00 equiv) was dissolved in 20 mL ofmethanol at 0° C. Concentrated sulfuric acid (4.3 mL) was addeddrop-wise and agitation was continued for 30 minutes at 0° C. Trimethylorthoformate (21.4 mL) was added, the cold bath removed and the reactionmedium left under agitation for 3 hours at ambient temperature. Thereaction medium was diluted with 200 mL of EtOAc, successively washedwith 100 mL of 10% Na₂CO₃ and 200 mL of saturated NaCl, then dried oversodium sulfate, filtered and concentrated under reduced pressure toyield 3.4 g (60%) of compound 1P in the form of a pale yellow oil.

Compound 1Q: [[1-(tert-butoxy)ethenyl]oxy](tert-butyl)dimethylsilane

Diisopropylamine (20 g, 186.71 m mol, 1.08 equiv) was dissolved in 170mL of THF in an inert atmosphere and cooled to −78° C. nBuLi (2.4 M,78.8 mL) was added drop-wise and the solution agitated 30 minutes at lowtemperature (to give LDA-lithium diisopropylamide) before addingtert-butyl acetate (20 g, 172.18 mmol, 1.00 equiv). The reaction mixturewas agitated 20 minutes at −78° C. before adding hexamethylphosphoramide(HMPA, 25.8 mL) and a solution of tertbutyldimethylchlorosilane(TBDMSCl, 28 g, 185.80 mmol, 1.08 equiv) in 35 mL of THF. Agitation wascontinued for 20 additional minutes at low temperature, and the coldbath was then removed. The solution was concentrated under reducedpressure. The residue was re-dissolved in 100 mL of water and extracted3 times with 100 mL of PE. The organic phases were combined, washed oncewith 500 mL of NaCl-saturated aqueous solution, dried over sodiumsulfate, filtered and concentrated. The residue was purified bydistillation to yield 16.6 g (83%) of compound 1Q in the form of acolourless oil.

Compound 1R: tert-butyl(3R,4S,5S)-4-[benzyl(methyl)amino]-3-methoxy-5-methyl heptanoate

Compound 1P (2.0 g, 7.54 mmol, 1.00 equiv) and compound 1Q (2.6 g, 11.28mmol, 1.50 equiv) were dissolved in 33 mL of DCM in an inert atmosphere.The solution was cooled to 0° C. DMF (1.2 g) was added drop-wisetogether with a solution of BF₃.Et₂O (2.1 g) in 7.5 mL of DCM. Agitationwas continued for 24 hours at 0° C. The reaction medium was washed oncewith 30 mL of sodium carbonate (10%) and twice with 50 mL ofNaCl-saturated aqueous solution, then dried over sodium sulfate,filtered and concentrated. The residue was purified on a silica columnwith a mixture of EtOAc and PE (1:100) to yield 1.82 g (91%) of compound1R in the form of a yellow oil.

Compound 1S: (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoatehydrochloride

Compound 1R (2.4 g, 6.87 mmol, 1.00 equiv) was dissolved in an inertatmosphere in 35 mL of ethanol in the presence of Pd/C (0.12 g) andconcentrated hydrochloric acid (0.63 mL). The nitrogen atmosphere wasreplaced by a hydrogen atmosphere and the reaction medium was left underagitation 18 hours at ambient temperature. The reaction medium wasfiltered and concentrated under reduced pressure. The residue wastriturated in 50 mL of hexane and the supernatant removed which, afterdrying under reduced pressure, gave 1.66 g (82%) of compound 1S in theform of a white solid.

Compound 1T: tert-butyl(3R,4S,5S)-4-[(2S)-2-[[(benzyloxy)carbonyl]amino]-N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoate

(2S)-2-[[(benzyloxy)carbonyl]amino]-3-methylbutanoic acid (15 g, 0.40mmol, 1.00 equiv) was dissolved in 300 mL of DCM in the presence of DIEA(38.3 mL) and bromotripyrrolidinophosphonium hexafluorophosphate(PyBrOP, 32.3 g). The solution was agitated 30 minutes at ambienttemperature before adding compound 1S (15.99 g, 0.42 mmol, 1.07 equiv).The reaction medium was agitated 2 hours and then concentrated. Theresidue was purified in reverse phase (C18) with a mixture ofacetonitrile (ACN) and water (30:70 to 100:0 in 40 minutes) to yield 17g (58%) of compound 1T in the form of a colourless oil.

Compound 1U: tert-butyl(3R,4S,5S)-4-[(2S)-2-amino-N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoate

Compound 1T (76 mg, 0.15 mmol, 1.00 equiv) was dissolved in an inertatmosphere in 10 mL of ethanol in the presence of Pd/C (0.05 g). Thenitrogen atmosphere was replaced by a hydrogen atmosphere and thereaction agitated 2 hours at ambient temperature. The reaction mediumwas filtered and concentrated under reduced pressure to yield 64 mg ofcompound 1U in the form of a colourless oil.

Compound 1V:(3R,4S,5S)-4-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoate

Compound 1U (18.19 g, 50.74 mmol, 1.00 equiv) was dissolved in 400 mL ofa 1,4-dioxane/water mixture (1:1) in the presence of sodium bicarbonate(12.78 g, 152 mmol, 3.00 equiv) and 9H-fluoren-9-ylmethyl chloroformate(Fmoc-C1, 19.69 g, 76 mmol, 1.50 equiv), then agitated 2 hours atambient temperature. The reaction medium was then diluted with 500 mL ofwater and extracted 3 times with 200 mL of EtOAc. The organic phaseswere combined, washed once with 200 mL of NaCl-saturated aqueoussolution, dried over sodium sulfate, filtered and concentrated to yield40 g of partly purified compound 1V in the form of a pale yellow oil.

Compound 1W:(3R,4S,5S)-4-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoicacid

Compound 1V (40 g, 68.88 mmol, 1.00 equiv) was dissolved in a neutralatmosphere in 600 mL of DCM. TFA (300 mL) was added. The solution wasagitated 2 hours at ambient temperature, then concentrated under reducedpressure. The residue was purified on a silica column with a mixture ofmethanol and DCM (1:10) to yield 23.6 g (65%) of compound 1W incolourless oil form.

Compound 1X: 9H-fluoren-9-ylmethylN-[(1S)-1-[[(3R,4S,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamoyl]ethyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl](methyl)carbamoyl]-2-methylpropyl] carbamate

Compound 1W (2.53 g, 4.82 mmol, 1.08 equiv) was dissolved in 20 mL ofDCM in the presence of compound 1K (2.18 g, 4.47 mmol, 1.00 equiv), DEPC(875 mg, 5.37 mmol, 1.20 equiv) and DIEA (1.25 g, 9.67 mmol, 2.16equiv). The reaction mixture was left under agitation overnight atambient temperature, then successively washed with 50 mL of saturatedKHSO₄ and 100 mL of water, dried over sodium sulfate, filtered andconcentrated. The residue was purified on a silica column with a mixtureof methanol and DCM (1:200 to 1:40) to yield 2.8 g (71%) of compound 1Xin the form of a pale yellow solid.

Compound 1Y:(2S)-2-amino-N-[(3R,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamoyl]ethyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl]-N,3-dimethylbutanamide

Compound 1X (2.8 g, 3.18 mmol, 1.00 equiv) was dissolved in acetonitrile(ACN, 12 mL) in the presence of piperidine (3 mL) and left underagitation 18 hours at ambient temperature. The reaction was neutralisedwith 50 mL of water, then extracted twice with 100 mL of DCM. Theorganic phases were combined, dried over sodium sulfate, filtered andconcentrated. The residue was purified on a silica column with a mixtureof methanol and DCM (1:100 to 1:40) to yield 1.2 g (57%) of compound 1Yin the form of a yellow solid.

Compound 1ZA: (2S)-2-[[(tert-butoxy)carbonyl](methyl)amino]-3-methylbutanoic acid

(2S)-2-[[(tert-butoxy)carbonyl]amino]-3-methylbutanoic acid (63 g,289.97 mmol, 1.00 equiv) was dissolved in an inert atmosphere in THF(1000 mL) in the presence of iodomethane (181 mL). The solution wascooled to 0° C. before adding sodium hydride (116 g, 4.83 mol, 16.67equiv) in small portions. The reaction mixture was agitated for 1.5hours at 0° C., the cold bath was then removed and agitation continuedfor 18 hours. The reaction was neutralised with 200 mL of water and thenconcentrated under reduced pressure. The residual aqueous phase wasdiluted with 4 litres of water, washed once with 200 mL of EtOAc and itspH adjusted to between 3 and 4 with a 1N solution of hydrochloric acid.The mixture obtained was extracted 3 times with 1.2 L of EtOAc. Theorganic phases were combined, dried over sodium sulfate, filtered andconcentrated to yield 60 g (89%) of compound 1ZA in the form of a yellowoil.

Compound 1ZB: benzyl(2S)-2-[[(tert-butoxy)carbonyl](methyl)amino]-3-methylbutanoate

Compound 1ZA (47 g, 203.21 mmol, 1.00 equiv) was dissolved in DMF (600mL) in the presence of Li₂CO₃ (15.8 g, 213.83 mmol, 1.05 equiv). Thesolution was cooled to 0° C. then benzyl bromide (BnBr 57.9 g, 338.53mmol, 1.67 equiv) was added drop-wise. The reaction mixture was leftunder agitation overnight before being neutralised with 400 mL of waterand filtered. The solution obtained was extracted twice with 500 mL ofEtOAc. The organic phases were combined, dried over sodium sulfate,filtered and concentrated. The residue was purified on a silica columnwith a mixture of EtOAc and PE (1:100 to 1:20) to yield 22.5 g (34%) ofcompound 1ZB in the form of a yellow oil.

Compound 1ZC: benzyl (2S)-3-methyl-2-(methylamino)butanoatehydrochloride

Compound 1ZB (22.5 g, 70.00 mmol, 1.00 equiv) was dissolved in 150 mL ofDCM. Gaseous hydrochloric acid was bubbled. The reaction was agitated 1hour at ambient temperature and then concentrated under reduced pressureto yield 17 g (94%) of compound 1ZC in the form of a yellow solid.

Compound 1ZD: tert-butyl N-(3,3-diethoxypropyl)carbamate

3,3-diethoxypropan-1-amine (6 g, 40.76 mmol, 1.00 equiv) was dissolvedin 1,4-dioxane (30 mL) in the presence of TEA (4.45 g, 43.98 mmol, 1.08equiv), then cooled to 0° C. (Boc)₂O (9.6 g, 43.99 mmol, 1.08 equiv)diluted in 20 mL of 1,4-dioxane was added drop-wise. The solution wasagitated 2 hours at 0° C. then overnight at ambient temperature beforebeing neutralised with 10 mL of water. The pH was adjusted to 5 with HCl(1%). The solution was extracted 3 times with 50 mL of EtOAc. Theorganic phases were combined, dried over sodium sulfate, filtered andconcentrated to yield 8.21 g (81%) of compound 1ZD in the form of a paleyellow oil.

Compound 1ZE: tert-butyl N-(3-oxopropyl) carbamate

Compound 1ZD (8.20 g, 33.15 mmol, 1.00 equiv) was dissolved in 18.75 mLof acetic acid and left under agitation overnight at ambienttemperature. The reaction medium was then extracted 3 times with 30 mLof EtOAc. The organic phases were combined, washed 3 times with 30 mL ofsaturated NaCl solution, dried over sodium sulfate, filtered andconcentrated to yield 5 g (87%) of compound 1ZE in the form of a darkred oil.

Compound 1ZF: (2S)-2-[(3-[[(tert-butoxy)carbonyl]amino]propyl)(methyl)amino]-3-methylbutanoic acid

Compound 1ZE (2.4 g, 13.86 mmol, 1.00 equiv) was dissolved in 50 mL ofTHF in the presence of compound 1ZC (3.56 g, 13.81 mmol, 1.00 equiv) andDIEA (9.16 mL, 4.00 equiv). The reaction mixture was agitated 30 minutesat ambient temperature before adding sodium triacetoxyborohydride (5.87g, 27.70 mmol, 2.00 equiv). Agitation was continued overnight, then thereaction was neutralised with 100 mL of water and extracted 3 times with50 mL of EtOAc. The organic phases were combined, dried over sodiumsulfate, filtered and concentrated. The residue was partly purified on asilica column with a mixture of EtOAc and PE (1:4). The crude productobtained was re-dissolved in 20 mL of methanol in the presence of Pd/C(1.2 g) and hydrogenated for 20 minutes at normal temperature andpressure. The reaction medium was filtered and concentrated underreduced pressure to yield 200 mg (5%) of compound 1ZF in the form of awhite solid.

Compound 1ZG: tert-butylN-(3-[[(1S)-1-[[(1S)-1-[[(3R,4S,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamoyl]thyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4yl](methyl)carbamoyl]-2-methylpropyl]carbamoyl]-2-methylpropyl](methyl)amino]propyl)carbamate

Compound 1Y (50 mg, 0.08 mmol, 1.00 equiv) was dissolved in 2 mL of DMFin the presence of compound 1ZF (26.2 mg, 0.09 mmol, 1.20 equiv), DIEA(37.7 mL) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 43.3 mg, 0.11 mmol, 1.50 equiv). The reactionwas left under agitation overnight at ambient temperature, then dilutedwith 10 mL of water and extracted 3 times with 5 mL of EtOAc. Theorganic phases were combined, dried over sodium sulfate, filtered andconcentrated to yield 100 mg of compound 1ZG in the form of a partlypurified colourless oil.

Compound 1ZG (90 mg, 0.10 mmol, 1.00 equiv) was dissolved in a neutralatmosphere in 2 mL of DCM and the solution was cooled with an ice bath.TFA (1 mL) was added and the reaction agitated for 2 hours at ambienttemperature, then concentrated under reduced pressure. The residue waspurified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN buffered with0.05% of TFA; Gradient of 18% to 31% ACN in 7 minutes then 31% to 100%ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220 nm).Compound 1 was obtained with a yield of 25% (23 mg) in the form of awhite solid.

LC/MS/UV (Atlantis T3 column, 3 μm, 4.6×100 mm; 35° C.; 1 mL/min, 30% to60% ACN in water (20 mM ammonium acetate in 6 minutes); ESI(C₄₄H₇₃N₇O₆S, exact masse 827.53) m/z: 829 (MH⁺), 5.84 min (93.7%, 254nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.85-7.80 (m,1H); 7.69-7.66 (m, 1H), 7.40-7.10 (m, 5H), 5.80-5.63 (m, 1H), 4.80-4.65(m, 2H), 4.22-4.00 (m, 1H), 3.89-0.74 (m, 58H).

Compound 2(S)-2-((S)-2-(((2-aminopyridin-4-yl)methyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 2A: tert-butyl(S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate

Compound 1D (2.5 g, 8.70 mmol, 1.00 equiv) and(1S,2R)-2-amino-1-phenylpropan-1-ol (1.315 g, 8.70 mmol, 1.00 equiv)were dissolved in an inert atmosphere in DMF (35 mL). The solution wascooled to 0° C. then DEPC (1.39 mL) and TEA (1.82 mL) were addeddrop-wise. The reaction mixture was agitated 2 hours at 0° C. then 4hours at ambient temperature. The reaction mixture was diluted with 200mL of water and extracted three times with 50 mL of EtOAc. The organicphases were combined, washed once with 50 mL of KHSO₄ (1 mol/L), oncewith 50 mL of NaHCO₃ (sat.), once with 50 mL of NaCl (sat.), then driedover sodium sulfate, filtered and concentrated under reduced pressure toyield 3.6 g (98%) of compound 2A in the form of a yellow solid.

Compound 2B:(2R,3R)—N-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide2,2,2-trifluoroacetate

Compound 2A (2.7 g, 6.42 mmol, 1.00 equiv) was dissolved in an inertatmosphere in DCM (40 mL) then cooled to 0° C. TFA (25 mL) was added andthe solution agitated for 2 hours at 0° C. The reaction mixture wasconcentrated under reduced pressure to yield 4.4 g of compound 2B in theform of a yellow oil.

Compound 2C: (9H-fluoren-9-yl)methyl((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

Compounds 2B (4.4 g, 10.13 mmol, 1.00 equiv) and 1W (5.31 g, 10.12 mmol,1.00 equiv) were dissolved in an inert atmosphere in DCM (45 mL). Thesolution was cooled to 0° C. then DEPC (1.62 mL) and DIEA (8.4 mL) wereadded drop-wise. The reaction mixture was agitated for 2 hours at 0° C.then at ambient temperature overnight. The reaction mixture was dilutedwith 100 mL of water and extracted three times with 50 mL of DCM. Theorganic phases were combined, washed once with 50 mL of KHSO₄ (1 mol/L),once with 50 mL of NaHCO₃ (sat.), once with 50 mL of NaCl (sat.), thendried over sodium sulfate, filtered and concentrated under pressure toyield 3.3 g (39%) of compound 2C in the form of a yellow solid.

Compound 2D:(S)-2-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide

Compound 2C (300 mg, 0.36 mmol, 1.00 eq.) was dissolved in an inertatmosphere in ACN (2 mL) and piperidine (0.5 mL). The solution was leftunder agitation at ambient temperature overnight then evaporated todryness under reduced pressure. The residue was purified on a silicacolumn with a mixture of DCM and MeOH (1:100) to yield 150 mg (68%) ofcompound 2D in the form of a white solid.

Compound 2E: methyl 2-((tert-butoxycarbonyl)amino)isonicotinate

Methyl 2-aminopyridine-4-carboxylate (2 g, 13.14 mmol, 1.00 equiv) wasdissolved in tert-butanol (20 mL) after which di-tert-butyl dicarbonate(4.02 g, 18.42 mmol, 1.40 equiv) was added. The reaction mixture wasagitated at 60° C. overnight then the reaction was halted through theaddition of an aqueous 1M NaHCO₃ solution (50 mL). The solid wasrecovered by filtration, washed with 50 mL of EtOH then dried in vacuoto yield 2.5 g (75%) of compound 2E in the form of a white solid.

Compound 2F: tert-butyl (4-(hydroxymethyl)pyridin-2-yl)carbamate

Compound 2E (2.5 g, 9.91 mmol, 1.00 equiv) and CaCl₂ (1.65 g) weredissolved in EtOH (30 mL). The solution was cooled to 0° C. then NaBH₄(1.13 g, 29.87 mmol, 3.01 equiv) was gradually added. The solution wasleft under agitation overnight at ambient temperature then the reactionwas halted with the addition of water (50 mL). The mixture was extractedthree times with 20 mL of EtOAc. The organic phases were combined,washed twice with 20 mL of NaCl (sat.) then dried over sodium sulfate,filtered and concentrated under reduced pressure to yield 2.0 g (90%) ofcompound 2F in the form of a colourless solid.

Compound 2G: tert-butyl (4-formylpyridin-2-yl)carbamate

Compound 2F (2.5 g, 11.15 mmol, 1.00 equiv) was dissolved in DCE (25 mL)then 19.4 g (223.14 mmol, 20.02 equiv) of MnO₂ were added. The mixturewas left under agitation overnight at 70° C. then the solids wereremoved by filtering. The filtrate was evaporated to dryness to yield1.4 g (57%) of compound 2G in the form of a white solid.

Compound 2H: benzyl(S)-2-(((2-((tert-butoxycarbonyl)amino)pyridin-4-yl)methyl)(methyl)amino)-3-methylbutanoate

Compound 2G (2.3 g, 10.35 mmol, 1.00 equiv) was dissolved in 25 mL ofTHF in the presence of compound 1ZC (2.93 g, 11.37 mmol, 1.10 equiv),DIEA (5.39 g, 41.71 mmol, 4.03 equiv) and NaBH(OAc)₃ (4.39 g, 20.71mmol, 2.00 equiv). The reaction mixture was agitated for 6 hours atambient temperature then neutralised with 60 mL of NaHCO₃ (sat.) andextracted 3 times with 20 mL of AcOEt. The organic phases were combined,washed twice with 20 mL of NaCl (sat.), dried over sodium sulfate,filtered and concentrated. The residue was purified on a silica columnwith a mixture of EtOAc and PE (1:15) to yield 2.7 g (61%) of compound2H in the form of a white solid.

Compound 2I: (S)-2-(((2-((tert-butoxycarbonyl)amino)pyridin-4-yl)methyl)(methyl)amino)-3-methylbutanoic acid

Compound 2H (500 mg, 1.17 mmol, 1.00 equiv) was dissolved in 10 mL ofAcOEt and 2 mL of methanol in the presence of Pd/C (250 mg), andhydrogenated for 3 hours at ambient temperature and atmosphericpressure. The reaction medium was filtered and concentrated underreduced pressure to yield 254 mg (64%) of compound 2I in the form of acolourless solid

Compound 2J: tert-butyl(4-((3S,6S,9S,10R)-9-((S)-sec-butyl)-10-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-3,6-diisopropyl-2,8-dimethyl-4,7-dioxo-11-oxa-2,5,8-triazadodecyl)pyridin-2-yl)carbamate

Compound 2J was prepared in similar manner to compound 1ZG from theamine 2D (85.2 mg, 0.14 mmol, 1.50 equiv), the acid 21 (31.7 mg, 0.09mmol, 1.00 equiv), HATU (42.9 mg, 0.11 mmol, 1.20 equiv) and DIEA (36.7mg, 0.28 mmol, 3.02 equiv) in DMF (3 mL). After evaporation to dryness,100 mg of crude product were obtained in the form of a white solid.

Compound 2J (100 mg, 0.11 mmol, 1.00 equiv) was dissolved in 2 mL of DCMand 1 mL of TFA. The reaction was agitated for 1 hour at ambienttemperature, then concentrated under reduced pressure. The residue (80mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFirePrep C18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN bufferedwith 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to100% ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220 nm).Compound 2 was obtained with a yield of 6% (6.3 mg) in the form of awhite solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.8mL/min, from 10% to 95% ACN in water (0.05% TFA) in 6 minutes); ESI(C₄₅H₇₃N₇O₇, exact mass 823.56) m/z: 824.5 (MH⁺) and 412.9 (M.2H⁺/2,100%), 3.21 min (99.2%, 210 nm)

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.81-7.79 (m,1H); 7.39-7.29 (m, 5H); 6.61-6.59 (m, 2H); 4.84-4.52 (m, 1H); 4.32-4.02(m, 1H); 3.90-2.98 (m, 10H); 2.90-2.78 (m, 1H); 2.55-0.81 (m, 39H).

Reference Compound 3 methyl((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

Compound 3A: tert-butyl(S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate

Compound 1D (3 g, 10.44 mmol, 1.00 equiv) and methyl(S)-2-amino-3-phenylpropanoate (2.25 g, 12.55 mmol, 1.20 equiv) weredissolved in an inert atmosphere in DMF (40 mL). The solution was cooledto 0° C. then DEPC (1.67 mL, 1.05 equiv) and TEA (3.64 mL, 2.50 equiv)were added drop-wise. The reaction mixture was agitated 2 hours at 0° C.then at ambient temperature overnight. The reaction mixture was dilutedwith 100 mL of water and extracted three times with 50 mL EtOAc. Theorganic phases were combined, washed once with 100 mL of KHSO₄ (1mol/L), once with 100 mL of NaHCO₃ (sat.), once with 100 mL of NaCl(sat.), then dried over sodium sulfate, filtered and concentrated underpressure to yield 4 g (85%) of compound 3A in the form of a colourlessoil.

Compound 3B: 2,2,2-trifluoroacetate of methyl(S)-2-((2R,3R)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamido)-3-phenylpropanoate

Compound 3A (5 g, 11.15 mmol, 1.00 equiv) was dissolved in an inertatmosphere in DCM (40 mL). TFA (25 mL) was added and the solutionagitated for 2 hours. The reaction mixture was concentrated underreduced pressure to yield 8 g of compound 3B in the form of a yellowoil.

Compound 3C: methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compounds 3B (8.03 g, 17.36 mmol, 1.00 equiv) and 1W (9.1 g, 17.34 mmol,1.00 equiv) were dissolved in an inert atmosphere in DCM (80 mL). Thesolution was cooled to 0° C. then DEPC (2.8 mL) and DIEA (12 mL) wereadded drop-wise. The reaction mixture was agitated for 2 hours at 0° C.then at ambient temperature overnight. The reaction mixture was dilutedwith 200 mL of water and extracted three times with 50 mL of DCM. Theorganic phases were combined, washed once with 50 mL of KHSO₄ (1 mol/L),once with 50 mL of NaHCO₃ (sat.), once with 50 mL of NaCl (sat.), thendried over sodium sulfate, filtered and concentrated under reducedpressure to yield 5 g (34%) of compound 3C in the form of a yellowsolid.

Compound 3D: methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-amino-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 3C (5.5 g, 6.43 mmol, 1.00 equiv) was dissolved in an inertatmosphere in a solution of tetrabutylammonium fluoride (TBAF, 2.61 g,9.98 mmol, 1.55 quiv) in DMF (100 mL). The solution was agitated atambient temperature for 2 hours then diluted with 100 mL of water andextracted three times with 50 mL of EtOAc. The organic phases werecombined then dried over sodium sulfate, filtered and concentrated underreduced pressure to yield 3.3 g (81%) of compound 3D in the form of ayellow solid.

Compound 3E: benzyl (S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanoate

Pyridine-4-carbaldehyde (1 g, 9.34 mmol, 1.00 equiv) was dissolved in 10mL of 1,2-dichloroethane (DCE) in the presence of compound 1ZC (2.9 g,11.25 mmol, 1.21 equiv) and titanium isopropoxide (IV) (4.19 mL, 1.40equiv). The mixture was agitated at ambient temperature for 30 minutesthen 2.77 g of NaBH(OAc)₃ (13.07 mmol, 1.40 equiv) were added. Thereaction medium was left under agitation overnight then neutralised with100 mL of water and the mixture extracted 3 times with 50 mL of AcOEt.The organic phases were combined and evaporated to dryness. The residuewas purified on a silica column with a mixture of EtOAc and PE (1:20) toyield 1.3 g (45%) of compound 3E in the form of a colourless oil.

Compound 3F: (S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanoicacid

Compound 3E (800 mg, 2.56 mmol, 1.00 equiv) was dissolved in 30 mL ofAcOEt in the presence of Pd/C (300 mg) and hydrogenated for 3 hours atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure. The residue waspurified on a silica column with a mixture of DCM and MeOH (100:1 to5:1) to yield 100 mg (18%) of compound 3F in the form of a white solid.

Compounds 3D (50 mg, 0.08 mmol, 1.00 equiv) and 3F (26.34 mg, 0.12 mmol,1.50 equiv) were dissolved in 3 mL of DCM. The solution was cooled to 0°C. then 0.018 mL of DEPC and 0.0392 mL of DIEA were added. The reactionwas agitated at 0° C. for 2 hours then at ambient temperature overnight.The reaction medium was concentrated under reduced pressure and theresidue (70 mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU,SunFire Prep C18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACNbuffered with 0.05% of TFA; Gradient of 20% to 40% ACN in 10 minutesthen 40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and220 nm). Compound 3 was obtained with a yield of 27% (20 mg) in the formof a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% ACN in water (0.05% TFA) in 8 minutes); ESI(C₄₆H₇₂N₆O₈, exact mass 836.5) m/z: 837.5 (MH⁺) and 419.4 (M.2H⁺/2(100%)), 7.04 min (90.0%, 210 nm)

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.76-8.74 (m,2H); 8.53-8.48 (m, 0.4H, NHCO incomplete exchange); 8.29-8.15 (m, 0.8H,NHCO incomplete exchange); 8.01 (s, 2H), 7.31-7.22 (m, 5H), 4.88-4.68(m, 3H); 4.31-4.07 (m, 2H); 3.94-2.90 (m, 18H); 2.55-0.86 (m, 38H).

Reference Compound 4(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, trifluoroacetic acid

Compound 3 (100 mg, 0.11 mmol, 1.00 equiv) was dissolved in a mixture ofwater (5 mL), ACN (5 mL) and piperidine (2.5 mL). The reaction mixturewas left under agitation overnight then concentrated under reducedpressure. The residue was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×150 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at254 nm and 220 nm), to yield 20 mg (20%) of compound 4 in the form of awhite solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% ACN in water (0.05% TFA) in 8 minutes); ESI(C₄₅H₇₀N₆O₈, exact mass 822.5) m/z: 823.5 (MH⁺) and 412.4 (M.2H⁺/2,100%), 6.84 min (89.1%, 210 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.79-8.78 (m,2H); 8.09 (m, 2H); 7.30-7.21 (m, 5H); 4.80-4.80 (m, 1H), 4.36-0.87 (m,58H).

Compound 6 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminopropyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 6A: methyl(2S)-2-[(2R)-2-[(R)-[(2S)-1-[(3R,4S,5S)-4-[(2S)-2-[(2S)-2-[(3-[[(tert-butoxy)carbonyl]amino]propyl)(methyl)amino]-3-methylbutanamido]-N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl](methoxy)methyl]propanamido]-3-phenylpropanoate

Compound 3D (157.5 mg, 0.25 mmol, 1.00 equiv) was dissolved at 0° C. inan inert atmosphere in 3 mL of DCM in the presence of carboxylic acid1ZF (78.7 mg, 0.27 mmol, 1.10 equiv), DEPC (46 μl) and DIEA (124 μl).The reaction mixture was agitated 2 hours at low temperature and thecold bath was then removed and agitation continued for 4 hours. It wasthen concentrated under reduced pressure to yield 200 mg of compound 6Ain the form of a crude yellow oil. It was used as such in the followingstep.

Compound 6A (200 mg, 0.22 mmol, 1.00 equiv) was dissolved in an inertatmosphere at 0° C. in 2 mL of DCM. TFA (1 mL) was added drop-wise andthe cold bath removed. The reaction mixture was agitated 1 hour atambient temperature then concentrated under reduced pressure. Theresidue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFirePrep C18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN bufferedwith 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to100% ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220 nm), toyield 60 mg (26%, yield in 2 steps) of compound 6 in the form of a whitesolid.

LC/MS/UV (Zorbax Eclipse Plus C8, 3.5 μm, 4.6×150 mm; 1 mL/min, 40° C.,30 to 80% methanol in water (0.1% H₃PO₄) in 18 minutes); ESI(C₄₃H₇₄N₆O₈, exact mass 802.56) m/z: 804 (MH⁺); 11.50 min (91.5%, 210nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.52 (d, 0.3H,NHCO incomplete exchange); 8.25 (d, 0.5H, NHCO incomplete exchange);7.30-7.22 (m, 5H); 4.9-4.6 (m, 3H); 4.2-4.0 (m, 1H); 4.0-0.86 (m, 61H).

Compound 7(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminopropyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid,bis trifluoroacetic acid

Compound 6 (70 mg, 0.08 mmol, 1.00 equiv) was dissolved in a mixture ofwater (5 mL), ACN (2.5 mL) and piperidine (5 mL). The reaction mixturewas left under agitation overnight at ambient temperature, thenconcentrated under reduced pressure. The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; UV Waters 2489 UV Detector at 254 nm and 220 nm), to yield 14.6mg (21%) of compound 7 in the form of a white solid.

LC/MS/UV (Ascentis Express C18, 2.7 μm, 4.6×100 mm; 1.5 mL/min, 40° C.,0 to 80% methanol in water (0.05% TFA) in 8 minutes); ESI (C₄₂H₇₂N₆O₈,exact mass 788.54) m/z: 790 (MH⁺), 5.71 min (96.83%, 210 nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.42 (d, 0.3H,NHCO incomplete exchange); 8.15 (d, 0.2H, NHCO incomplete exchange);7.31-7.21 (m, 5H); 4.9-4.6 (m, 3H); 4.25-4.0 (m, 1H); 4.0-0.86 (m, 59H).

Compound 8(S)-2-((S)-2-(((2-aminopyridin-4-yl)methyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 8A: tert-butyl(4-((3S,6S,9S,10R)-9-((S)-sec-butyl)-3,6-diisopropyl-10-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-2,8-dimethyl-4,7-dioxo-11-oxa-2,5,8-triazadodecyl)pyridin-2-yl)carbamate

Compound 8A was synthesised in the same manner as for compound 2J fromthe amine 1Y (39 mg, 0.06 mmol, 1.00 equiv), the acid 21 (20 mg, 0.06mmol, 1.00 equiv), HATU (27 mg, 0.07 mmol, 1.20 equiv) and DIEA (23.2mg, 0.18 mmol, 3.01 equiv) in DCM (3 mL). The crude product was notpurified.

Compound 8: Compound 8 was synthesised in similar manner to compound 2from the intermediate 8A (100 mg, 0.10 mmol, 1.00 equiv). The crudeproduct (100 mg) was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×150 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 18% to 31% ACN in 7minutes then 31% to 100% ACN in 2 minutes; Waters 2489 UV Detector at254 nm and 220 nm). Compound 8 was obtained with a yield of 8% (8 mg) inthe form of a white solid.

LC/MS/UV (Atlantis T3 column, 3 μm, 4.6×100 mm; 35° C.; 1.8 mL/min, 25%to 80% ACN in water (0.05% TFA) in 7 minutes); ESI (C₄₇H₇₂N₈O₆S, exactmass 876.5) m/z: 877.5 (MH⁺) and 439.5 (M.2H⁺/2, 100%), 4.87 min (95.1%,254 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.83-7.78 (m,2H); 7.56-7.52 (m, 1H); 7.34-7.17 (m, 5H); 6.64-6.62 (m, 2H); 5.77-5.61(m, 1H); 4.86-4.68 (m, 2H); 4.25-4.05 (m, 1H); 3.87-2.83 (m, 17H);2.56-0.84 (m, 37H).

Compound 9 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(((2-aminopyridin-4-yl)methyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

Compound 9A: methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(((2-((tert-butoxycarbonyl)amino)pyridin-4-yl)methyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 9A was synthesised in the same manner as for compound 3 fromthe amine 3D (170 mg, 0.27 mmol, 1.00 equiv), the acid 21 (99.7 mg, 0.30mmol, 1.10 equiv), DEPC (0.049 mL, 1.05 equiv) and DIEA (0.133 mL, 3.00equiv) in DCM (5 mL). The crude product was purified on a silica columnwith a mixture of EtOAc and PE (1:1) to yield 200 mg (78%) of compound9A in the form of a pale yellow solid.

Compound 9: Compound 9 was synthesised in the same manner as forcompound 2 from the intermediate 9A (200 mg, 0.21 mmol, 1.00 equiv) inDCM (4 mL) and TFA (2 mL). The crude product was purified by preparativeHPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×150mm; Eluting phase: water/ACN buffered with 0.05% TFA; Gradient of 20% to40% ACN in 10 minutes then 40% to 100% ACN in 2 minutes; Waters 2545 UVDetector at 254 nm and 220 nm). Compound 9 was obtained with a yield of10% (20 mg) in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₆H₇₃N₇O₈, exact mass 851.6) m/z: 852.5 (MH⁺) and 426.9 (M.2H⁺/2,100%), 6.92 min (92.7%, 254 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.51-8.45 (m,0.5H, NH incomplete exchange); 8.30-8.24 (m, 0.3H, NH incompleteexchange); 8.17-8.07 (m, 0.8H, NH incomplete exchange); 7.79-7.77 (m,1H); 7.36-7.18 (m, 5H); 7.21-7.16 (m, 1H); 6.94-6.89 (m, 1H); 4.85-4.65(m, 3H); 4.20-3.10 (m, 20H); 3.00-2.85 (m, 2H); 2.55-0.80 (m, 36H).

Compound 10(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(((2-aminopyridin-4-yl)methyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, trifluoroacetic acid

Compound 10: Compound 9 (100 mg, 0.11 mmol, 1.00 equiv) was dissolved ina mixture of water (5 mL), ACN (5 mL) and piperidine (2.5 mL). Thereaction mixture was left under agitation overnight at ambienttemperature and then concentrated under reduced pressure. The residuewas purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire PrepC18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN buffered with0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACNin 2 minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield32.2 mg (33%) of compound 10 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₅H₇₁N₇O₆, exact mass 837.5) m/z: 838.5 (MH⁺) and 419.9 (M.2H⁺/2,100%), 6.81 min (97.7%, 220 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.41-8.32 (m,0.3H, NH incomplete exchange); 8.20-8.07 (m, 0.8H, NH incompleteexchange); 7.82-7.75 (m, 1H); 7.36-7.158 (m, 5H); 7.12-7.03 (m, 1H);6.94-6.88 (m, 1H); 4.85-4.66 (m, 3H); 4.20-3.10 (m, 16H); 3.00-2.85 (m,2H); 2.57-0.80 (m, 37H).

Compound 11(S)—N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamide, trifluoroacetic acid

Compound 11A: tert-butyl N-[4-(2-hydroxyethyl)phenyl]carbamate

Di-tert-butyl dicarbonate (16.7 g, 77 mmol, 1.05 eq.) was added to asolution of 2-(4-aminophenyl)ethanol (10 g, 72.9 mmol, 1 eq.) in THF(200 mL), and the reaction stirred overnight at ambient temperature. Themixture was diluted with EtOAc (200 mL), washed with water (200 mL),then HCl 1M (100 mL), then saturated aqueous NaHCO₃ solution (100 mL)then brine (100 mL). The organic phase was dried over MgSO₄ thenevaporated to dryness under reduced pressure. The crude product wastriturated twice with heptane (150 mL) and dried under vacuum to furnishcompound 11A as a white solid (14.7 g, 84%).

Compound 11B: tert-butyl N-[4-(2-oxoethyl)phenyl]carbamate

Compound 11A (2.5 g, 10.5 mmol, 1.00 equiv) was dissolved in 25 mL ofDCM then cooled to −78° C. A Dess-Martin Periodinane solution (DMP, 6.71g, 15.8 mmol, 1.5 equiv) in DCM (10 mL) was added drop-wise. The coldbath was removed and agitation continued for 1 hour at ambienttemperature. The reaction was neutralised with 60 mL of a 50/50 mixtureof sodium bicarbonate-saturated aqueous solution and Na₂S₂O₃-saturatedaqueous solution. The resulting solution was extracted 3 times with 30mL of EtOAc. The organic phases were combined, washed twice withNaCl-saturated aqueous solution, dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure. The residue waspurified on silica gel (EtOAc/PE 1/15) to yield 1.0 g (40%) of compound11B in the form of a pale yellow solid.

Compound 11C: benzyl (2S)-2-[[2-(4-[[(tert-butoxy)carbonyl]amino]phenyl)ethyl] (methyl)amino]-3-methylbutanoate

Compound 1ZC (3.5 g, 13.6 mmol, 1.1 equiv) was dissolved in THF (30 mL)in the presence of DIEA (6.4 g, 49.7 mmol, 4.0 equiv), aldehyde 11B (2.9g, 12.3 mmol, 1.0 equiv) and sodium triacetoxyborohydride (5.23 g, 49.7mmol, 2.0 equiv). The reaction mixture was left under agitationovernight at ambient temperature, then neutralised with 60 mL of sodiumbicarbonate-saturated solution. The resulting solution was extracted 3times with 30 mL EtOAc. The organic phases were combined, washed twicewith NaCl-saturated aqueous solution, dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified on silica gel (EtOAc/PE 1:20) to yield 3.7 g (68%) ofcompound 11C in the form of a yellow oil.

Compound 11D:(2S)-2-[[2-(4-[[(tert-butoxy)carbonyl]amino]phenyl)ethyl](methyl)amino]-3-methylbutanoicacid

Compound 11C (2 g, 4.5 mmol, 1 equiv) was dissolved in 10 mL of methanolin the presence of Pd/C (2 g) and hydrogenated for 2 hours at normaltemperature and pressure. The reaction medium was filtered andconcentrated under reduced pressure to yield 1.2 g (75%) of compound 11Din the form of a yellow oil.

Compound 11E:(2S)-2-[[2-(4-[[(tert-butoxy)carbonyl](methyl)amino]phenyl)ethyl](methyl) amino]-3-methylbutanoic acid

Compound 11D (1.2 g, 3.4 mmol, 1.00 equiv) was dissolved in an inertatmosphere in THF (20 mL). The reaction medium was cooled with an icebath after which NaH (60% in oil, 549 mg, 13.7 mmol, 4.0 equiv) wasadded in portions, followed by iodomethane (4.9 g, 34 mmol, 10 equiv).The reaction was left under agitation overnight at ambient temperature,then neutralised with water and washed with 100 mL of EtOAc. The pH ofthe aqueous solution was adjusted to 6-7 with 1N HCl. This aqueoussolution was extracted 3 times with 100 mL of EtOAc. The organic phaseswere combined, dried over sodium sulfate, filtered and concentrated toyield 800 mg (64%) of compound 11E in the form of a yellow solid.

Compound 11F: tert-butylN-[4-(2-[[(1S)-1-[[(1S)-1-[[(3R,4S,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamoyl]ethyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4yl](methyl)carbamoyl]-2-methylpropyl]carbamoyl]-2-methylpropyl](methyl)amino]ethyl)phenyl]-N-methylcarbamate

Compound 11F was prepared in similar manner to compound 6A from theamine 1Y (150 mg, 0.22 mmol, 1.2 equiv) and the acid 11E (70 mg, 0.19mmol, 1.0 equiv). After purification on silica gel (EtOAc/PE 1:1) 100 mg(52%) of desired product were obtained in the form of a pale yellowsolid.

Compound 11 was prepared in the same manner as for compound 1 from theintermediate 11F (100 mg, 0.1 mmol). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2489 UV Detector at 254 nm and 220 nm). Compound 11 wasobtained with a yield of 39% (39.7 mg) in the form of a white solid.

LC/MS/UV (Eclipse Plus C8, 3.5 μm, 4.6×150 mm; 1 mL/min, 40° C., 50 to95% methanol in water (0.05% TFA) in 18 minutes); ESI (C₅₀H₇₇N₇O₆S,exact mass 903.57) m/z: 904.5 (MH⁺), 7.53 min (93.68%, 254 nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.84 (d, 0.5H,NHCO incomplete exchange); 8.7-8.5 (m, 0.9H, NHCO incomplete exchange);7.76-7.73 (m, 1H); 7.55-7.4 (m, 1H); 7.28-7.22 (m, 7H); 7.08-7.05 (m,2H); 5.51-5.72 (m, 1H); 4.9-4.80 (m, 2H); 4.3-0.7 (m, 60H).

Compound 12 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

In the same manner as for the final phases in the synthesis of compound1, compound 12 was prepared in two steps from the amine 3D (118 mg, 0.19mmol) and the acid 11E (82 mg, 0.22 mmol). The final residue waspurified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN buffered with0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACNin 2 minutes; Waters 2489 UV Detector at 254 nm and 220 nm). Compound 12was obtained with a yield of 7% (13.7 mg) in the form of a white solid.

LC/MS/UV (Eclipse Plus C8, 3.5 μm, 4.6×150 mm; 1 mL/min, 40° C., 40 to95% methanol in water (0.05% TFA) in 18 minutes); ESI (C₄₉H₇₈N₆O₈, exactmass 878.59) m/z: 879.7 (MH⁺), 10.07 min (90.6%, 254 nm).

¹H:NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.40 (se, 2H);7.38-7.22 (m, 7H); 4.95-4.7 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 62H).

Compound 13(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, trifluoroacetic acid

Compound 13 was prepared in the same manner as for compound 7 fromcompound 12 (100 mg, 0.10 mmol). The residue was purified by preparativeHPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×150mm; Eluting phase: water/ACN buffered with 0.05% TFA; Gradient of 20% to40% ACN in 10 minutes then 40% to 100% ACN in 2 minutes; Waters 2489 UVDetector at 254 nm and 220 nm). Compound 13 was obtained with a yield of20% (20 mg) in the form of a white solid.

LC/MS/UV (Ascentis Express C18, 2.7 μm, 4.6×100 mm; 1.5 mL/min, 40° C.,10 to 95% methanol in water (0.05% TFA) in 8 minutes); ESI (C₄₈H₇₆N₆O₈,exact mass 864.57) m/z: 865.6 (MH⁺), 6.05 min (90.9%, 210 nm).

¹H NMR: (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.32-7.19 (m,9H); 4.9-4.65 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 59H).

Compound 14(S)-2-((S)-2-((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 14A: tert-butyl (3-(hydroxymethyl)phenyl) carbamate

(3-aminophenyl)methanol (3 g, 24.36 mmol, 1.00 equiv) was dissolved inTHF (60 mL) after which di-tert-butyl dicarbonate (6.38 g, 29.23 mmol,1.20 equiv) was then added. The reaction mixture was left underagitation overnight at ambient temperature and the reaction was thendiluted by adding 200 mL of water. The product was extracted 3 timeswith 100 mL of AcOEt and the organic phases were then recombined, driedover sodium sulfate, filtered and concentrated under reduced pressure toyield the crude product (13.85 g of compound 14A) in the form of ayellow oil.

Compound 14B: tert-butyl (3-formylphenyl)carbamate

Compound 14A (13.8 g, 61.81 mmol, 1.00 equiv) was dissolved in DCE (400mL) and MnO₂ (54 g, 621.14 mmol, 10.05 equiv) was then added. Themixture was left under agitation at ambient temperature for 3 days afterwhich the solids were removed by filtering. The filtrate was evaporatedto dryness and the residue was purified on a silica column with amixture of EtOAc and PE (1:30) to yield 3 g (22%) of compound 14B in theform of a white solid.

Compound 14C: benzyl (S)-2-((3-((tert-butoxycarbonyl)amino)benzyl)(methyl)amino)-3-methylbutanoate

Compound 14B (1 g, 4.52 mmol, 1.00 equiv) was dissolved in 20 mL of THFin the presence of compound 1ZC (1.16 g, 4.50 mmol, 1.00 equiv), DIEA (3mL) and NaBH(OAc)₃ (1.92 g, 9.06 mmol, 2.01 equiv). The reaction mixturewas left under agitation overnight at ambient temperature and thenneutralised with 100 mL of water and extracted 3 times with 50 mL ofAcOEt. The organic phases were combined, dried over sodium sulfate,filtered and concentrated. The residue was purified on a silica columnwith a mixture of EtOAc and PE (1:50) to yield 1.9 g (99%) of compound14C in the form of a white solid.

Compound 14D: (S)-2-((3-((tert-butoxycarbonyl)amino)benzyl)(methyl)amino)-3-methylbutanoic acid

Compound 14C (1 g, 2.34 mmol, 1.00 equiv) was dissolved in 30 mL ofAcOEt and 4 mL of methanol in the presence of Pd/C (400 mg) andhydrogenated for 1 hour at ambient temperature and atmospheric pressure.The reaction medium was filtered and concentrated under reduced pressureto yield 680 mg (86%) of compound 14D in the form of a white solid.

Compound 14E: tert-butyl (3-((3S,6S,9S,1OR)-9-((S)-sec-butyl)-3,6-diisopropyl-10-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-2,8-dimethyl-4,7-dioxo-11-oxa-2,5,8-triazadodecyl)phenyl)carbamate

Compound 14E was synthesised in the same manner as for compound 3 fromthe amine 1Y (100 mg, 0.15 mmol, 1.00 equiv), the acid 14D (102.27 mg,0.30 mmol, 2.00 equiv), DEPC (0.053 mL) and DIEA (0.046 mL) in DCM (3mL). The crude product (80 mg) was purified on a silica column with amixture of EtOAc and PE (1:1) to yield 100 mg (67%) of compound 14E inthe form of a pale yellow solid.

Compound 14 was synthesised in the same manner as for compound 2 fromthe intermediate 14E (100 mg, 0.10 mmol, 1.00 equiv). The crude product(80 mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFirePrep C18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN bufferedwith 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to100% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220 nm).Compound 14 was obtained with a yield of 10% (10 mg) in the form of awhite solid.

LC/MS/UV (Eclipse plus C8 column, 3.5 μm, 4.6×150 mm; 40° C.; 1.0mL/min, 40% to 95% MeOH in water (0.05% TFA) in 18 minutes); ESI(C₄₈H₇₃N₇O₆S, exact mass 875.5) m/z: 876.5 (M^(H)) and 438.9 (M.2H/2,100%), 11.35 min (95.6%, 210 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.92-8.86 (m,0.4H, NH incomplete exchange); 8.70-8.54 (m, 0.6H, NH incompleteexchange); 7.88-7.78 (m, 1H); 7.60-7.50 (m, 1H); 7.45-6.97 (m, 9H);5.80-5.65 (m, 1H); 4.85-4.70 (m, 1H); 4.40-0.80 (m, 56H).

Compound 15 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

Compound 15A: methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-((tert-butoxycarbonyl)amino)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 15A was synthesised in the same manner as for compound 3 fromthe amine 3D (200 mg, 0.32 mmol, 1.00 equiv), the acid 14D (212.6 mg,0.63 mmol, 2.00 equiv), DEPC (0.1103 mL) and DIEA (0.157 mL, 3.00 equiv)in DCM (5 mL). The crude product was purified on a silica column with amixture of EtOAc and PE (1:1) to yield 200 mg (67%) of compound 15A inthe form of a yellow solid.

Compound 15 was synthesised in the same manner as for compound 2 fromthe intermediate 15A (200 mg, 0.21 mmol, 1.00 equiv). The crude productwas purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire PrepC18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACN buffered with0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACNin 2 minutes; Waters UV Detector 2545 at 254 nm and 220 nm). Compound 15was obtained with a yield of 19% (38.6 mg) in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₇H₇₄N₆O₈, exact mass 850.5) m/z: 851.5 (MH⁺) and 426.4 (M.2H⁺/2,100%), 6.61 min (91.1%, 210 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.53-7.42 (m,1H); 7.35-7.18 (m, 8H); 4.88-4.79 (m, 2H); 4.42-4.00 (m, 3H); 3.93-2.71(m, 22H); 2.61-0.81 (m, 33H).

Compounds 16 to 20

Compounds 16 to 20 were prepared in the same manner as for compound 1,from the amines 1Y and 1ZC and corresponding aldehydes.

The tert-butyl (4-oxobutyl)carbamate, involved in the preparation ofcompound 16, was prepared as for compound 1ZE in 2 steps from4,4-diethoxybutan-1-amine.

The tert-butyl-N-methyl-N-(2-oxoethyl)carbamate involved in thepreparation of compound 17 was commercial.

The 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetaldehyde, involved inthe preparation of compound 18, was prepared in 2 steps as follows:

2-(2-Hydroxyethoxy)ethan-1-ol (7 g, 66 mmol, 9.9 equiv) was dissolved inan inert atmosphere in pyridine (10 mL) in the presence of4-dimethylaminopyridine (DMAP, 80 mg, 0.65 mmol, 0.1 equiv). Thesolution was cooled to 0° C. then TBDMSCl (1 g, 6.6 mmol, 1.0 equiv) wasadded in portions. The reaction mixture was left under agitationovernight at ambient temperature, diluted with 100 mL of EtOAc andsuccessively washed twice with 100 mL of 1N HCl and twice withNaCl-saturated aqueous solution. The organic phase was dried overanhydrous sodium sulphate, filtered and concentrated under reducedpressure to yield 1.3 g (88%) of2-[2-[(tert-butyldimethylsilyl)oxy]ethoxy]ethan-1-ol in the form of acolourless oil.

The oxalyl chloride (760 mg, 6 mmol, 1.3 equiv) was dissolved in aninert atmosphere in DCM (40 mL) and cooled to −78° C. Dimethylsulfoxide(DMSO, 1.07 g, 13.7 mmol, 3 equiv) diluted in DCM (5 mL) was addeddrop-wise. After an agitation time of 30 minutes at low temperature,2-[2-[(tert-butyldimethyl silyl)oxy]ethoxy]ethan-1-ol (1 g, 4.5 mmol,1.0 equiv) dissolved in 5 mL of DCM was added. Agitation was continuedfor 1 hour at low temperature before adding TEA (2.78 g, 27 mmol, 6equiv). The reaction mixture was agitated 15 minutes at −78° C. andovernight at ambient temperature before being neutralised with 100 mL ofwater. It was then extracted 3 times with 100 mL of DCM. The organicphases were combined, dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified on silicagel (EtOAc/PE 1:20) and yielded 0.8 g (80%) of2-[2-[(tert-butyldimethylsilyl)oxy]ethoxy]acetaldehyde in the form of acolourless oil.

The tert-butyl 4-formylphenyl carbonate involved in the preparation ofcompound 19 was prepared in a single step as follows:4-hydroxybenzaldehyde (2.5 g, 20.5 mmol, 1.0 equiv) was dissolved in aninert atmosphere in THF (20 mL) in the presence of 18-crown-6 (0.25 g)and potassium carbonate (5 g). The reaction mixture was cooled to 0° C.and the di-tert-butyl dicarbonate (5.8 g, 26.58 mmol, 1.30 equiv) wasthen added. Agitation was continued for 1 hour at low temperature afterwhich the reaction was neutralised with 30 mL of water. The resultingsolution was extracted three times with 200 mL of EtOAc. The organicphases were combined, dried over anhydrous sodium sulfate filtered andconcentrated under reduced pressure. The residue was purified on silicagel (EtOAc/PE 1:10) and yielded 4.2 g (92%) of tert-butyl 4-formylphenylcarbonate in the form of a pale yellow solid.

The 4-nitrobenzaldehyde involved in the preparation of compound 20 wascommercial.

The synthesis of compound 18 was completed by deprotection of thesilylated alcohol. This was performed as follows:(S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-11-isopropyl-2,2,3,3,10-pentamethyl-4,7-dioxa-10-aza-3-siladodecan-12-amide(40 mg, 0.04 mmol, 1.0 equiv) was dissolved in an inert atmosphere inTHF (10 mL) in the presence of TBAF (2 mg, 0.09 mmol, 2 equiv) andagitated 4 hours at ambient temperature. The reaction was neutralisedwith 50 mL of water then extracted three times with 50 mL of EtOAc. Theorganic phases were combined, dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to yield compound 18 inthe crude state.

The synthesis of compound 20 was completed by reducing the nitro group.This was performed as follows:(2S)—N-[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl]-1-methoxy-2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N,3-dimethyl-2-[(2S)-3-methyl-2-[methyl[(4-nitrophenyl)methyl]amino]butanamido]butanamide (40 mg, 0.05 mmol, 1.0 equiv) was dissolvedin 15 mL of ethanol. Dihydrated tin chloride (II) (317 mg, 1.4 mmol, 30equiv) was added and the solution left under agitation for 3 days atambient temperature. The reaction was neutralised with 50 mL of water,then extracted three times with 50 mL of EtOAc. The organic phases werecombined, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to yield compound 20 in the crude state.

No Name x R Purity* Quantity 16 (S)-2-((S)-2-((4-aminobutyl)(methyl)amino)-3- methylbutanamido)-N- ((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2- 2

94.9% 11.5 mg methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino) propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide, bis trifluoroaceticacid 17 (S)-N-((3R,4S,5S)-3-methoxy-1- ((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl- 1-(thiazol-2-yl)ethyl)amino) 2

99.6% 65.5 mg propyl)pyrrolidin-1-yl)-5- methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2- (methyl(2-(methylamino)ethyl)amino)butanamido)butanamide, bis trifluoroacetic acid 18(S)-2-((S)-2-((2-(2- hydroxyethoxy)ethyl)(methyl)amino)-3-methylbutanamido)-N- ((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2- 1

94.5% 46.4 mg methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino) propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide, trifluoroacetic acid19 (S)-2-((S)-2-((4-hydroxybenzyl) (methyl)amino)-3-methylbutanamido)-N- ((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2- methyl-3-oxo-3-(((S)-2-phenyl- 1

93.2% 21.6 mg 1-(thiazol-2-yl)ethyl)amino) propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide, trifluoroacetic acid20 (S)-2-((S)-2-((4-aminobenzyl) (methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3- methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3- (((S)-2-phnyl-1-(thiazol-2- 1

96.7% 21.1 mg yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4- yl)-N,3-dimethylbutanamide,trifluoroacetic acid *The compounds were purified by preparative HPLC(Pre-HPLC-001 SHIMADZU, SUNFIRE ™ Prep C18 OBD column, 5 μm, 19 × 150mm; Eluting phase: water/ACN buffered with 0.05% TFA; Gradient of 20% to40% ACN in 10 minutes then 40% to 100% ACN in 2 minutes; Waters 2489 UVDetector at 254 nm and 220 nm), to give the corresponding TFA salts inthe form of white solids.

Characterization of the end products: Compound 16 LC/MS/UV (Eclipse PlusC8, 3.5 μm, 4.6×150 mm; 1 mL/min, 40° C., 5 to 95% methanol in water(0.05% TFA) in 18 minutes); ESI: (C₄₅H₇₅N₇O₆S, exact mass 841.55) m/z842.5 (MH⁺), 421.9 (100%, (M.2H⁺)/2); UV: 14.02 min (94.9%, 210 nm). ¹HNMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.55-8.2 (m, 0.8H,NHCO incomplete exchange); 8.0 (0.55H, NHCO incomplete exchange); 7.70(d, 1H); 7.44 (d, 1H); 7.21-7.15 (m, 5H); 5.65-5.45 (m, 1H); 4.8-4.5 (m,2H); 4.15-3.9 (m, 2H); 3.8-0.6 (m, 59H). Compound 17 LC/MS/UV ESI:(C₄₄H₇₃N₇O₆S, exact mass 827.53) m/z 828 (MH⁺), 415 [100%, (M.2H⁺)/2];UV: RT=6.72 min (99.6%, 254 nm)¹H NMR: (400 MHz, CD₃OD, ppm): δ(Presence of rotamers) 7.82-7.80 (m, 1H); 7.56-7.54 (m, 1H); 7.35-7.20(m, 5H); 5.8-5.55 (m, 1H); 4.85-4.6 (m, 1H); 4.25-4.05 (m, 1H); 3.95-0.8(m, 60H). Compound 18 LC/MS/UV (Atlantis T3, 3 μm, 4.6×100 mm; 1.2mL/min, 40° C., 5 to 95% methanol in water (0.05% TFA) in 7 minutes) i;ESI: (C₄₅H₇₄N₆O₈S, exact mass 858.53) m/z 859 (MH⁺), 881 (MNa⁺), 430(100%, (M.2H⁺)/2); UV: 4.85 min (96.8%, 220 nm). ¹H NMR: (400 MHz,CD₃OD, ppm): δ (Presence of rotamers) 8.75-8.55 (m, 0.5H, NHCOincomplete exchange); 7.85-7.80 (m, 1H); 7.6-7.5 (m, 1H); 7.40-7.15 (m,5H); 5.8-5.6 (m, 1H); 4.8-4.55 (m, 2H); 4.15-4.0 (m, 1H); 4.0-0.8 (m,60H). Compound 19 LC/MS/UV ESI: (C₄₈H₇₂N₆O₇S, exact mass 876.52) m/z 877(MH⁺), 439 [100%, (M.2H⁺)/2]; UV: RT=1.76 min (93.2%, 220 nm). Compound20 ¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.85-7.80 (m,1H); 7.6-7.5 (m, 1H); 7.4-7.15 (m, 5H); 7.1-7.05 (m, 2H); 6.73-6.70 (m,2H); 5.8-5.55 (m, 1H); 5.0-4.7 (m, 2H); 4.25-4.05 (m, 1H); 4.0-0.8 (m,54H). LC/MS/UV ESI: (C₄₈H₇₃N₇O₇S, exact mass 875.53) m/z 876 (MH⁺), 439[75%, (M.2H⁺)/2]; UV: RT=4.83 min (96.8%, 254 nm). ¹H NMR (400 MHz,CD₃OD, ppm): δ (Presence of rotamers) 7.85-7.80 (m, 1H); 7.6-7.5 (m,1H); 7.4-7.1 (m, 7H); 6.76-6.72 (m, 2H); 5.8-5.55 (m, 1H); 4.9-4.65 (m,2H); 4.25-4.05 (m, 1H); 4.0-0.8 (m, 54H).

Compounds 21 to 24

Compounds 21 to 24 were prepared in the same manner as for compounds 17to 20, replacing the amine 1Y by the amine 2D.

No Name x R Purity* Quantity 21 (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl- 3-oxopropyl)pyrrolidin-1-yl)-3- 2

97.5% 24.4 mg methoxy-5-methyl-1-oxoheptan- 4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2- (methylamino)ethyl)amino) butanamido)butanamide, bistrifluoroacetic acid 22 (S)-N-(3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl- 3-oxopropyl)pyrrolidin-1-yl)-3- 1

95.5% 26.1 mg methoxy-5-methyl-1-oxoheptan- 4-yl)-2-((S)-2-((2-(2-hydroxyethoxy)ethyl)(methyl) amino)-3-methylbutanamido)-N,3-dimethylbutanamide, trifluoroacetic acid 23(S)-N-(3R,4S,5S)-1-((S)-2- ((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2- yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan- 1

98.5%  5.8 mg 4-yl)-2-((S)-2-((4- hydroxybenzyl)(methyl)amino)-3-methylbutanamido)-N,3- dimethylbutanamide, trifluoroacetic acid 24(S)-2-((S)-2-((4-aminobenzyl) (methyl)amino)-3- methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1- 1

99.1%  6.9 mg methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan- 4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid *The compounds were purified by preparative HPLC(Pre-HPLC-001 SHIMADZU, SUNFIRE ™ Prep C18 OBD column, 5 μm, 19 × 150mm; Eluting phase: water/ACN buffered with 0.05% TFA; Gradient of 20% to40% ACN in 10 minutes then 40% to 100% ACN in 2 minutes; Waters 2489 UVDetector at 254 nm and 220 nm), to give the corresponding TFA salts inthe form of white solids.

Characterization of the end products: Compound 21 LC/MS/UV (ESI)(C₄₂H₇₄N₆O₇, exact mass 774.56) m/z 775 (MH⁺), 797 (MNa⁺), 388 (100%,(M.2H⁺)/2); UV: 3.14 min (97.6%, 215 nm). ¹H NMR (400 MHz, CD₃OD, ppm):δ (Presence of rotamers) 8.05-7.7 (m, 0.8H, NHCO incomplete exchange);7.45-7.15 (m, 5H); 4.9-4.45 (m, 2H); 4.35-4.00 (m, 2H); 3.95-0.8 (m,61H). Compound 22 LC/MS/UV (ESI) (C₄₃H₇₅N₅O₉, exact mass 805.56) m/z 806(MH⁺), 828 (MNa⁺), 404 (100%, (M.2H⁺)/2); UV: 4.47 min (95.6%, 215 nm).¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.1-7.7 (m,0.4H, NHCO incomplete exchange); 7.45-7.15 (m, 5H); 4.9-4.5 (m, 3H);4.4-4.05 (m, 2H); 4.05-0.8 (m, 61H). Compound 23 LC/MS/UV (ESI)(C₄₆H₇₃N₅O₈, exact mass 823.55) m/z 824 (MH⁺), 846 (MNa⁺), 413 (100%,(M.2H+)/2); UV: 4.76 min (98.5%, 215 nm). ¹H NMR (400 MHz, CDCl₃, ppm):δ (Presence of rotamers) 7.5-7.2 (m, 5H); 7.9-7.75 (m, 2H); 5.5-5.3 (m,1H); 4.9-4.6 (m, 2H); 4.55-4.15 (m, 4H); 4.0-0.8 (m, 55H). Compound 24LC/MS/UV (ESI) (C₄₆H₇₄N₆O₇, exact mass 822.56) m/z 823 (MH⁺), 845(MNa⁺), 861 (MK⁺); UV: 3.68 min (99.15%, 254 nm). ¹H NMR (400 MHz,CD₃OD, ppm): δ (Presence of rotamers) 8.0-7.7 (m, 0.5H, NHCO incompleteexchange); 7.5-7.0 (m, 7H); 6.75-6.65 (m, 2H); 4.85-4.5 (m, 2H);4.4-4.05 (m, 2H); 4.0-0.8 (m, 56H).

Compound 26(S)-2-((S)-2-((2-aminoethyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,bis trifluoroacetic acid

Compound 26A: benzyl(S)-2-((2-((tert-butoxycarbonyl)amino)ethyl)(methyl)amino)-3-methylbutanoate

Compound 26A was prepared in the same manner as for compound 2H from theamine 1ZC (1.3 g, 5.04 mmol, 1.00 equiv), tert-butyl(2-oxoethyl)carbamate (800 mg, 5.03 mmol, 1.00 equiv), DIEA (3.52 g,27.24 mmol, 5.42 equiv) and NaBH(OAc)₃ (2.25 g, 10.62 mmol, 2.11 equiv)in THF (25 mL). The mixture was left under agitation overnight andneutralised with 50 mL of water. The residue was purified on a silicacolumn with a mixture of EtOAc and PE (10:1) to yield 0.6 g (33%) ofcompound 26A in the form of a colourless oil.

Compound 26B:(S)-2-((2-((tert-butoxycarbonyl)amino)ethyl)(methyl)amino)-3-methylbutanoicacid

Compound 26A (600 mg, 1.65 mmol, 1.00 equiv) was dissolved in 40 mL ofTHF in the presence of Pd/C (300 mg) and hydrogenated for 1 hour atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure. The residue waspurified on a silica column with a mixture of EtOAc and MeOH to yield0.4 g (89%) of compound 26B in the form of a colourless oil.

Compound 26C: tert-butyl((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)carbamate

Compound 26C was prepared in the same manner as for compound 3 from theamine 1Y (70 mg, 0.11 mmol, 1.00 equiv), the acid 26B (58.4 mg, 0.21mmol, 2.00 equiv), DEPC (0.032 mL) and DIEA (0.053 mL) in DCM (3 mL).After evaporation to dryness, compound 26C was obtained in the form of ayellow oil (100 mg).

Compound 26: Compound 26 was synthesised in the same manner as forcompound 2 from the intermediate 26C (100 mg, 0.11 mmol, 1.00 equiv) inDCM (3 mL) and TFA (1.5 mL). The crude product was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 45% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm). Compound 26 wasobtained with a yield of 38% (38.1 mg) in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.0mL/min, 5% to 95% MeOH in water (0.05% TFA) on 18 minutes); ESI(C₄₃H₇₁N₇O₆S, exact mass 813.52) m/z: 814.5 (MH⁺) and 407.9 (M.2H⁺/2,100%), 15.78 min (91.2%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.90-8.82 (m,0.5H, NH incomplete exchange); 8.71-8.65 (m, 0.3H, NH incompleteexchange); (7.85-7.77 (m, 1H); 7.60-7.49 (m, 1H); 7.37-7.15 (m, 5H);5.78-5.55 (m, 1H); 4.82-4.62 (m, 1.6H); 4.32-3.83 (m, 3.6H); 3.75-3.35(m, 7.4H); 3.30-2.60 (m, 13H); 2.58-0.80 (m, 42H).

Compound 27 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

Compound 27: Compound 27 was prepared in the same manner as for compound3 from the amine 3D (70 mg, 0.11 mmol, 1.00 equiv), the acid 49C (55.5mg, 0.22 mmol, 2.00 equiv), DEPC (0.034 mL, 2.00 equiv) and DIEA (0.055mL, 3.00 equiv) in DCM (3 mL). The crude product was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 45% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm). Compound 27 wasobtained with a yield of 3% (2.9 mg) in the form of a white solid.

LC/MS/UV (Eclipse Plus C8 column, 3.5 μm, 4.6×150 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₈H₇₅N₅O₉, exact mass 866.56) m/z: 866.5 (MH⁺) and 433.9 (M.2H⁺/2,100%), 6.61 min (89.1%, 210 nm).

¹H NMR (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.70-8.49 (m,0.9H, NH/OH incomplete exchange); 8.30-8.22 (m, 0.3H, NH incompleteexchange); 7.36-7.02 (m, 7H); 6.86-6.62 (m, 2H); 4.82-4.69 (m, 2H);4.20-4.03 (m, 1H); 3.91-3.33 (m, 12H); 3.30-2.90 (m, 17H); 2.55-0.80 (m,35H).

Compound 28(S)-2-((S)-2-((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 28A: tert-butyl(3-((3S,6S,9S,10R)-9-((S)-sec-butyl)-10-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-3,6-diisopropyl-2,8-dimethyl-4,7-dioxo-11-oxa-2,5,8-triazadodecyl)phenyl)carbamate

Compound 28A was prepared in the same manner as for compound 3 from theamine 2D (100 mg, 0.17 mmol, 1.00 equiv), the acid 14D (111.25 mg, 0.33mmol, 2.00 equiv), DEPC (0.058 mL) and DIEA (0.05 mL) in DCM (3 mL). Theresidue was purified on a silica column with a mixture of EtOAc andhexane (1:1) to yield 100 mg (66%) of compound 28A in the form of awhite solid.

Compound 28: Compound 28 was synthesised in the same manner as forcompound 2 from the intermediate 28A (100 mg, 0.11 mmol, 1.00 equiv).The crude product (80 mg) was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×150 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at254 nm and 220 nm). Compound 28 was obtained with a yield of 20% (20 mg)in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₆H₇₄N₆O₇, exact mass 822.56) m/z: 823.5 (MH⁺) and 412.4 (M.2H⁺/2,100%), 12.45 min (87.2%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.47-7.20 (m,5H); 7.10-7.01 (m, 1H); 6.80-6.56 (m, 3H); 4.82-4.52 (m, 3H); 4.33-4.03(m, 2H); 3.91-3.82 (m, 0.5H); 3.75-3.35 (m, 9.5H); 3.28-3.10 (m, 2H);2.79-2.90 (m, 1H); 2.60-2.40 (m, 2H); 2.30-0.80 (m, 40H).

Compound 29(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, trifluoroacetic acid

Compound 15 (100 mg, 0.10 mmol, 1.00 equiv) was dissolved in a mixtureof water (5 mL), ACN (5 mL) and piperidine (2.5 mL). The reactionmixture was left under agitation overnight at ambient temperature andthen concentrated under reduced pressure. The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 20 mg(20%) of compound 29 in the form of a white solid.

LC/MS/UV (Eclipse Plus C8 column, 3.5 μm, 4.6×150 mm; 40° C.; 1.0mL/min, 40% to 95% MeOH in water (0.05% TFA) in 18 minutes); ESI(C₄₆H₇₂N₆O₈, exact mass 836.54) m/z: 837.5 (MH⁺) and 419.4 (M.2H⁺/2,100%), 10.61 min (92.5%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.38-7.15 (m,6H); 7.00-6.99 (m, 3H); 4.85-4.68 (m, 2H); 4.37-3.38 (m, 11H); 3.31-2.70(m, 8H); 2.60-0.82 (m, 35H).

Compound 30 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-aminobutyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 30 was prepared in the same manner as for compound 16, from theamine 3D and the corresponding carboxylic acid.

LC/MS/UV (Ascentis Express C18, 2.7 μm, 4.6×100 mm; 1.5 mL/min, 40° C.,10 to 95% methanol in water (0.05% TFA) in 15 minutes); ESI (C₄₄H₇₆N₆O₈,exact mass 816.57) m/z: 817.6 (MH⁺), 409.4 (M.2H⁺/2); 12.0 min (90%, 210nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.7-8.2 (m, 1H,NHCOs, incomplete exchange); 7.4-7.1 (m, 5H); 4.95-4.7 (m, 3H); 4.2-4.0(m, 1H); 3.9-0.8 (m, 63H).

Compound 31(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-aminobutyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, bis trifluoroacetic acid

Compound 31 was prepared in the same manner as for compound 4, from themethyl ester 30.

LC/MS/UV (Ascentis Express C18, 2.7 μm, 4.6×100 mm; 1.5 mL/min, 40° C.,10 to 95% methanol in water (0.05% TFA) in 18 minutes); ESI (C₄₃H₇₄N₆O₈,exact mass 802.56) m/z: 803.6 (MH⁺), 402.4 (M.2H⁺/2); 13.68 min (98.9%,210 nm).

¹H NMR (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.4-7.1 (m, 5H);4.95-4.7 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.8 (m, 61H).

Compound 32(S)—N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(3-(methylamino)propyl)amino)butanamido)butanamide, bis trifluoroacetic acid

Compound 32A: tert-butyl (3,3-diethoxypropyl)(methyl)carbamate

Compound 1ZD (247 mg, 1 mmol, 1.00 equiv) was dissolved in an inertatmosphere in 30 mL of a 1:1 mixture of THF and DMF. The reaction mediumwas cooled over an ice bath after which the NaH (60% in oil, 60 mg, 1.5equiv) was added in portions, followed by the MeI (0.28 mL) drop-wise.The reaction was left under agitation for 2 days at ambient temperature,then neutralised with 5 mL of NH₄Cl-saturated aqueous solution andextracted twice with 15 mL of EtOAc. The organic phases were combined,dried over sodium sulfate, filtered and concentrated under reducedpressure to yield 200 mg (77%) of compound 32A in the form of a yellowsolid.

Compound 32B: (S)-2-((3-((tert-butoxycarbonyl)(methyl)amino)propyl)(methyl)amino)-3-methylbutanoic acid

Compound 32B was prepared following the same protocol described for thepreparation of compound 1ZF, replacing compound 1ZD by compound 32A.

Compound 32: Compound 32 was prepared in two steps, following the sameprotocol described for the preparation of compound 1, from the amine 1Yand the carboxylic acid 32B.

LC/MS/UV (Zorbax SB-Aq, 1.8 μm, 4.6×100 mm, 40° C., 10 to 95% methanolin water (0.05% TFA) in 13 minutes); ESI (C₄₅H₇₅N₇O₆S, exact mass842.19) m/z: 843 (MH⁺), 421.9 (M.2H⁺/2); 11.91 min (88%, 210 nm).

¹H NMR: (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.5-9.0 (m,0.5H, incomplete exchange NHCOs), 7.85-7.80 (m, 1H); 7.60-7.50 (m, 1H),7.35-7.15 (m, 5H), 5.80-5.63 (m, 1H), 4.80-4.65 (m, 2H), 4.30-4.00 (m,1H), 3.95-0.80 (m, 61H).

Compounds 33 and 34

Compounds 33 and 34 were prepared in the same manner as for compounds 6and 7, replacing the carboxylic acid 1ZF by compound 32B.

N° Name R Purity* Quantity 33 methyl (S)-2-((2R,3R)-3-((S)-1- Me 95% 32mg ((7S,10S,13S,14R)-13-((S)-sec-butyl)-7,10-diisopropyl-14-methoxy-6,12-dimethyl-8,11-dioxo-2,6,9,12-tetraazahexadecan-16- oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate, bis trifluoroacetic acid 34(S)-2-((2R,3R)-3-((S)-1-((7S,10S,13S,14R)- H 98% 18 mg13-(S)-sec-butyl)-7,10-diisopropyl-14-methoxy-6,12-dimethyl-8,11-dioxo-2,6,9,12-tetraazahexadecan-16-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3- phenylpropanoic acid, bistrifluoroacetic acid *The compounds were purified by preparative HPLC(Pre-HPLC-001 SHIMADZU, SUNFIRE ™ Prep C18 OBD column, 5 μm, 19 × 150mm; Eluting phase: water/ACN buffered with 0.05% TFA; Gradient of 20% to40% ACN in 10 minutes then 40% to 100% ACN in 2 minutes; Waters 2489 UVDetector at 254 nm and 220 nm), to yield the corresponding TFA salts inthe form of white solids.

Characterization of the end products: Compound 33 LC/MS/UV (ESI)(C₄₄H₇₆N₆O₈, exact mass 816.57) m/z 817.6 (MH⁺), 409.4 (M.2H⁺/2); UV:5.94 min (95%, 210 nm). ¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence ofrotamers) δ 8.6-8.2 (m, 0.8H, NHCO incomplete exchange) 7.30-7.22 (m,5H), 4.80 (m, 2H), 4.23-0.86 (m, 66H). Compound 34 LC/MS/UV (ESI)(C₄₃H₇₄N₆O₈, exact mass 802.56) m/z 803.6 (MH⁺), 402.4 (M.2H⁺/2); UV:5.94 min (97.5%, 210 nm). ¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence ofrotamers) δ 7.30-7.20 (m, 5H), 4.80 (m, 2H), 4.25-0.86 (m, 63H).

Compound 35(S)-2-((S)-2-((2-(2-aminoethoxy)ethyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,bis trifluoroacetic acid

Compound 35A: tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate

2-(2-aminoethoxy)ethanol (5 g, 47.56 mmol, 1.00 equiv) was dissolved inTHF (100 mL) at 0° C. and sodium hydroxide (2 g, 50.00 mmol, 1.05 equiv)was then added (solution in 25 mL of water). A solution of di-tert-butyldicarbonate (10.38 g, 47.56 mmol, 1.00 equiv) in THF (20 mL) was addeddrop-wise and the reaction was then left under agitation overnight atambient temperature. The reaction was diluted by adding 50 mL of waterand the product was extracted with 3 times 75 mL of AcOEt. The organicphases were combined, washed once with 100 mL of NaCl (sat.), then driedover sodium sulfate, filtered and concentrated under reduced pressure toyield 9 g (92%) of compound 35A in the form of a yellow oil.

Compound 35B: tert-butyl (2-(2-oxoethoxy)ethyl)carbamate

A solution of DMSO (3.46 mL, 5.00 equiv) in DCM (20 mL) was addeddrop-wise to a solution of oxalyl chloride (1.9 mL, 2.30 equiv) in DCM(20 mL) at −78° C. under nitrogen. After the addition (30 min), thesolution was agitated for 30 minutes and a solution of compound 35A (2g, 9.74 mmol, 1.00 equiv) in 20 mL DCM was then added. After theaddition of TEA (12.2 mL), the reaction was agitated at −78° C. for 30minutes and then at ambient temperature overnight. The reaction wasdiluted by adding 100 mL of water and the product was extracted 3 timeswith 50 mL of AcOEt. The organic phases were combined, dried over sodiumsulfate, filtered and concentrated under reduced pressure to yield 1.9 gof compound 35B in the form of a yellow oil.

Compound 35C: benzyl(S)-12-isopropyl-2,2,11-trimethyl-4-oxo-3,8-dioxa-5,11-diazatridecan-13-oate

Compound 35C was synthesised in the same manner as for compound 14C fromthe amine 1ZC (2.4 g, 9.31 mmol, 1.00 equiv), the aldehyde 35B (1.9 g,9.35 mmol, 1.00 equiv), NaBH(OAc)₃ (3.96 g, 18.68 mmol, 2.00 equiv) andDIEA (6.2 mL) in THF (40 mL). The reaction mixture was neutralised with200 mL of water and extracted 3 times with 100 mL of AcOEt. The organicphases were combined, dried over sodium sulfate, filtered andconcentrated to yield 2.3 g of compound 35C in the form of a yellow oil.

Compound 35D:(S)-12-isopropyl-2,2,11-trimethyl-4-oxo-3,8-dioxa-5,11-diazatridecan-13-oicacid

Compound 35C (200 mg, 0.49 mmol, 1.00 equiv) was dissolved in 10 mL ofEtOH in the presence of Pd/C (200 mg) and hydrogenated overnight. Thereaction medium was filtered and concentrated under reduced pressure toyield 150 mg (96%) of compound 35D in the form of a white solid.

Compound 35E: tert-butyl ((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2,14-dioxa-5,8,11-triazahexadecan-16-yl)carbamate

Compound 35E was synthesised in the same manner as for compound 3 fromthe amine 1Y (70 mg, 0.11 mmol, 1.00 equiv), the acid 35D (40.6 mg, 0.13mmol, 1.20 equiv), DEPC (0.0324 mL) and DIEA (0.0527 mL) in DCM (3 mL).The crude product (100 mg, 98%) was isolated in the form of a yellow oiland subsequently used as such.

Compound 35: Compound 35 was synthesised in the same manner as forcompound 2 from the intermediate 35E (100 mg, 0.10 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-010),SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase: water/ACNbuffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220nm). Compound 35 was obtained with a yield of 23% (22.9 mg) in the formof a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₅H₇₅N₇O₇S, exact mass 857.54) m/z: 858.5 (MH⁺) and 429.9 (M.2H⁺/2,100%), 5.89 min (89.7%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) δ 8.9-8.5 (m,0.5H, NHCO incomplete exchange), 7.8-7.7 (m, 1H), 7.55-7.45 (m, 1H),7.35-7.1 (m, 5H), 5.45-5.5 (m, 1H), 4.9-4.6 (m, 1H), 4.3-0.75 (m, 62H).

Compound 36 methyl(S)-2-((2R,3R)-3-((S)-1-((7S,10S,13S,14R)-1-amino-13-((S)-sec-butyl)-7,10-diisopropyl-14-methoxy-6,12-dimethyl-8,11-dioxo-3-oxa-6,9,12-triazahexadecan-16-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 36A: methyl(S)-2-((2R,3R)-3-((S)-1-((12S,15S,18S,19R)-18-((S)-sec-butyl)-12,15-diisopropyl-19-methoxy-2,2,11,17-tetramethyl-4,13,16-trioxo-3,8-dioxa-5,11,14,17-tetraazahenicosan-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 36A was synthesised in the same manner as for compound 3 fromthe amine 3D (50 mg, 0.08 mmol, 1.00 equiv), the acid 35D (25 mg, 0.11mmol, 1.48 equiv), DEPC (0.0337 mL) and DIEA (0.0548 mL) in DCM (3 mL).The crude product (100 mg) was isolated in the form of a yellow oil andsubsequently used as such.

Compound 36: Compound 36 was synthesised in the same manner as forcompound 2 from the intermediate 36A (100 mg, 0.11 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at254 nm and 220 nm). Compound 36 was obtained with a yield of 13% (12.7mg) in the form of a white solid.

LC/MS/UV (Agilent ZORBAX SB-Aq column, 1.8 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 2% MeOH in water (0.05% TFA) for 1 minute then 2% to 95% MeOH inwater in 13 minutes, then 95% MeOH in water for 2 minutes); ESI(C₄₄H₇₆N₆O₉, exact mass 832.57) m/z: 833.5 (MH⁺) and 417.4 (M.2H⁺/2,100%), 11.58 min (98.5%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.1-8.5 (m,0.8H, NHCO incomplete exchange), 7.30-7.1 (m, 5H), 4.9-4.6 (m, 3H),4.2-0.8 (m, 64H).

Compound 37(S)-2-((2R,3R)-3-((S)-1-((7S,10S,13S,14R)-1-amino-13-((S)-sec-butyl)-7,10-diisopropyl-14-methoxy-6,12-dimethyl-8,11-dioxo-3-oxa-6,9,12-triazahexadecan-16-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, bis trifluoroacetic acid

Compound 37 was prepared in the same manner as for compound 4, fromcompound 36 (100 mg, 0.11 mmol, 1.00 equiv). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 18.4 mg(19%) of compound 37 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₃H₇₄N₆O₉, exact mass 818.6) m/z: 819.6 (MH⁺) and 410.4 (M.2H⁺/2,100%), 5.48 min (96.7%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.35-7.2 (m,5H), 5.0-4.65 (m, 3H), 4.3-0.8 (m, 61H).

Compound 38 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((2-aminoethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 38A: methyl(S)-2-((2R,3R)-3-((S)-1-((9S,12S,15S,16R)-15-((S)-sec-butyl)-9,12-diisopropyl-16-methoxy-2,2,8,14-tetramethyl-4,10,13-trioxo-3-oxa-5,8,11,14-tetraazaoctadecan-18-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 38A was synthesised in the same manner as for compound 3 fromthe amine 3D (70 mg, 0.11 mmol, 1.00 equiv), the acid 26B (60.7 mg, 0.22mmol, 2.00 equiv), DEPC (0.0337 mL) and DIEA (0.0548 mL) in DCM (3 mL).The crude product (100 mg) was isolated in the form of a yellow oil.

Compound 38: Compound 38 was synthesised in the same manner as forcompound 2 from the intermediate 38A (100 mg, 0.11 mmol, 1.00 equiv).The crude product was purified by preparative HPLCPre-HPLC-001 SHIMADZU,SunFire Prep C18 OBD column, 5 μm, 19×150 mm; Eluting phase: water/ACNbuffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then40% to 100% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220nm). Compound 38 was obtained with a yield of 10% (10.3 mg) in the formof a white solid.

LC/MS/UV (Agilent ZORBAX SB-Aq column, 1.8 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 2% MeOH in water (0.05% TFA) for 1 minute then 2% to 95% MeOH inwater in 13 minutes then 95% MeOH in water for 2 minutes); ESI(C₄₂H₇₂N₆O₈, exact mass 788.5) m/z: 789.5 (MH⁺) and 395.4 (M.2H⁺/2,100%), 12.97 min (91.0%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.30-7.1 (m,5H), 4.9-4.6 (m, 3H), 4.2-0.8 (m, 60H).

Compound 39(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((2-aminoethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, bis trifluoroacetic acid

Compound 39 was prepared in the same manner as for compound 4, fromcompound 38 (100 mg, 0.11 mmol, 1.00 equiv). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 8.2 mg(8%) of compound 39 in the form of a white solid.

LC/MS/UV (Eclipse Plus C18 column, 3.5 μm, 4.6×150 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₁H₇₀N₆O₈, exact mass 774.5) m/z: 775.5 (MH⁺) and 388.4 (M.2H⁺/2,100%), 6.47 min (93.6%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.35-7.15 (m,5H), 4.9-4.6 (m, 3H), 4.2-0.8 (m, 57H).

Compound 40(S)—N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)butyl)amino)butanamido)butanamide,bis trifluoroacetic acid

Compound 40A: tert-butyl (4,4-diethoxybutyl)(methyl)carbamate

Compound 40A was prepared in the same manner as for compound 11E, fromtert-butyl(4,4-diethoxybutyl)(methyl)carbamate (5.5 g, 19.97 mmol, 1.00equiv), NaH (60% in oil, 3.2 g, 80.00 mmol, 4.01 equiv) and iodomethane(14 mL) in THF/DMF (40/20 mL). The reaction was neutralised with 50 mLof NH₄Cl(aq). Compound 40A was isolated in the form of a yellow oil, 5.5g (95%).

Compound 40B: tert-butyl methyl(4-oxobutyl)carbamate

Compound 40A (3 g, 10.89 mmol, 1.00 equiv) was dissolved in a mixture ofAcOH and water (15/4 mL) and the solution was left under agitationovernight. The pH was brought to 7-8 with NaHCO₃-saturated aqueoussolution and then extracted twice with 50 mL of EtOAc. The organicphases were combined, washed twice with 50 mL of NaCl-saturated aqueoussolution, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. Compound 40B was isolated in the form of yellowoil, 2.1 g (96%).

Compound 40C: benzyl(S)-2-((4-((tert-butoxycarbonyl)(methyl)amino)butyl)(methyl)amino)-3-methylbutanoate

Compound 40C was synthesised in the same manner as for compound 14C fromthe amine 1ZC (2.45 g, 9.53 mmol, 0.80 equiv), the aldehyde 40B (2.4 g,11.92 mmol, 1.00 equiv), NaBH(OAc)₃ (5.06 g, 23.87 mmol, 2.00 equiv) andDIEA (6.16 g, 47.66 mmol, 4.00 equiv) in THF (15 mL). The reactionmixture was neutralised with 100 mL of water and extracted twice with100 mL of AcOEt. The organic phases were combined, dried over sodiumsulfate, filtered and concentrated. The residue was purified on silicagel (EtOAc/PE (1:100-1:20)) to yield 1.2 g (25%) of compound 40C in theform of a yellow oil.

Compound 40D: (S)-2-((4-((tert-butoxycarbonyl)(methyl)amino)butyl)(methyl) amino)-3-methylbutanoic acid

Compound 40C (500 mg, 1.23 mmol, 1.00 equiv) was dissolved in 20 mL ofEtOH in the presence of Pd/C (550 mg) and hydrogenated for 1 hour atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure to yield 350 mg (90%)of compound 40D in the form of a colourless oil.

Compound 40E: tert-butyl((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-yl)(methyl)carbamate

Compound 40E was synthesised in the same manner as for compound 3 fromthe amine 1Y (60 mg, 0.09 mmol, 1.00 equiv), the acid 40D (57.8 mg, 0.18mmol, 2.00 equiv), DEPC (0.0278 mL) and DIEA (0.0452 mL) in DCM (3 mL).The crude product (100 mg) was subsequently used as such.

Compound 40: Compound 40 was synthesised in the same manner as forcompound 2 from the intermediate 40E (100 mg, 0.10 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×100 mm Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254nm and 220 nm). Compound 40 was obtained with a yield of 24% (24.6 mg)in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₆H₇₇N₇O₆S, exact mass 855.6) m/z: 856.6 (MH⁺) and 428.8 (M.2H⁺/2,100%), 5.89 min (97.0%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.9-8.5 (0.7H,NHCO incomplete exchange), 7.8-7.7 (m, 1H), 7.55-4.45 (m, 1H), 7.35-7.1(m, 5H), 5.5-5.75 (m, 1H), 4.9-4.6 (m, 2H), 4.2-0.8 (m, 64H).

Compound 41 methyl(S)-2-((2R,3R)-3-((S)-1-((8S,11S,14S,15R)-14-((S)-sec-butyl)-8,11-diisopropyl-15-methoxy-7,13-dimethyl-9,12-dioxo-2,7,10,13-tetraazaheptadecan-17-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 41A: methyl(S)-2-((2R,3R)-3-((S)-1-((11S,14S,17S,18R)-17-((S)-sec-butyl)-11,14-diisopropyl-18-methoxy-2,2,5,10,16-pentamethyl-4,12,15-trioxo-3-oxa-5,10,13,16-tetraazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 41A was synthesised in the same manner as for compound 3 fromthe amine 3D (170 mg, 0.27 mmol, 1.00 equiv), the acid 40D (170 mg, 0.54mmol, 2.09 equiv), DEPC (0.0819 mL) and DIEA (0.133 mL) in DCM (5 mL).The crude product (200 mg) was subsequently used as such.

Compound 41: Compound 41 was synthesised in the same manner as forcompound 2 from the intermediate 41A (100 mg, 0.11 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254nm and 220 nm). Compound 41 was obtained with a yield of 25% (25 mg) inthe form of a white solid.

LC/MS/UV (Agilent Zorbax SB-Aq column, 1.8 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 2% MeOH in water (0.05% TFA) for 1 minute then 2% to 95% MeOH inwater in 13 minutes, then 95% MeOH in water for 2 minutes); ESI(C₄₅H₇₈N₆O₈, exact mass 830.6) m/z: 831.6 (MH⁺) and 416.4 (M.2H⁺/2,100%), 11.58 min (97.2%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.55-8.15(0.75H, NHCO incomplete exchange), 7.30-7.1 (m, 5H), 4.9-4.6 (m, 3H),4.2-0.8 (m, 67H).

Compound 42(S)-2-((2R,3R)-3-((S)-1-((8S,11S,14S,15R)-14-((S)-sec-butyl)-8,11-diisopropyl-15-methoxy-7,13-dimethyl-9,12-dioxo-2,7,10,13-tetraazaheptadecan-17-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, bis trifluoroacetic acid

Compound 42 was prepared in the same manner as for compound 4, fromcompound 41 (100 mg, 0.11 mmol, 1.00 equiv). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 30.6 mg(31%) of compound 42 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 0% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₄H₇₆N₆O₈, exact mass 816.6) m/z: 817.6 (MH⁺) and 409.4 (M.2H⁺/2,100%), 5.75 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.5-8.1 (0.3H,NHCO incomplete exchange), 7.30-7.1 (m, 5H), 4.9-4.6 (m, 3H), 4.2-0.8(m, 64H).

Compound 43(S)—N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((R)-3-methyl-2-(methyl(2-(2-(methylamino)ethoxy)ethyl)amino)butanamido)butanamide, bis trifluoroacetic acid

Compound 43A: tert-butyl(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)ethyl) carbamate

Compound 35A (tert-butyl ((2-(2-hydroxyethoxy)ethyl)carbamate) (8.21 g,40.00 mmol, 1.00 equiv) and imidazole (6 g, 88.14 mmol, 2.20 equiv) weredissolved in an inert atmosphere in DCM (200 mL).Tertbutyldimethylsilane chloride (TBDMSCl, 6.62 g, 43.92 mmol, 1.10equiv) was added drop-wise and the reaction medium was left underagitation overnight at ambient temperature. The reaction mixture wasdiluted with 100 mL of DCM then washed twice with 200 mL of 0.5 M HCl,twice with 200 mL of NaHCO₃ (sat.), then 300 mL of NaCl (sat.). Theorganic phase was dried over sodium sulfate, filtered and concentratedunder reduced pressure. The residue was purified on silica gel (EtOAc/PE(1:3) to yield 10 g (78%) of compound 43A in the form of a white solid.

Compound 43B: tert-butyl (2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)ethyl)(methyl)carbamate

Compound 43B was prepared in the same manner as for compound 11E, fromcompound 43A (10 g, 31.30 mmol, 1.00 equiv), NaH (60% in oil, 5 g,208.33 mmol, 4.00 equiv) and iodomethane (22 g, 5.00 equiv) in DMF (200mL). The reaction medium was neutralised with 200 mL of water and washed3 times with 100 mL of AcOEt then 300 mL of NaCl (sat.). The organicphase was dried over sodium sulfate, filtered and concentrated underreduced pressure to yield 10 g (96%) of compound 43B in the form of awhite solid.

Compound 43C: tert-butyl (2-(2-hydroxyethoxy)ethyl)(methyl)carbamate

Compound 43B (10 g, 29.89 mmol, 1.00 equiv) and TBAF.3H₂O (20.8 g, 65.93mmol, 2.20 equiv) were dissolved in THF (200 mL). The mixture wasagitated at ambient temperature for 2 hours then extracted 3 times with100 mL of AcOEt. The organic phases were recombined, washed twice with300 mL of water, then twice with 300 mL of NaCl (sat.), dried oversodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified on silica gel (EtOAc/PE (1:3-1:1) to yield 6.6 g ofcompound 43C in the form of colourless oil.

Compound 43D: tert-butyl methyl(2-(2-oxoethoxy)ethyl)carbamate

Compound 43D was prepared in the same manner as for compound 35B, fromcompound 43C (2 g, 9.12 mmol, 1.00 equiv), oxalyl chloride (1.9 mL), TEA(11.3 mL) and DMSO (3.3 mL). Compound 43D (2 g) was isolated in the formof yellow oil.

Compound 43E: benzyl(S)-12-isopropyl-2,2,5,11-tetramethyl-4-oxo-3,8-dioxa-5,11-diazatridecan-13-oate

Compound 43E was synthesised in the same manner as for compound 14C fromthe amine 1ZC (2.4 g, 9.31 mmol, 1.00 equiv), the aldehyde 43D (2 g,9.16 mmol, 1.00 equiv), NaBH(OAc)₃ (4 g, 18.87 mmol, 2.06 equiv) andDIEA (6 mL) in THF (100 mL). The reaction mixture was neutralised with100 mL of water and extracted 3 times with 100 mL of AcOEt. The organicphases were combined, dried over sodium sulfate, filtered andconcentrated. The residue was purified on silica gel (EtOAc/PE (4:1) toyield 1 g (37%) of compound 43E in the form of a white solid.

Compound 43F:(S)-12-isopropyl-2,2,5,11-tetramethyl-4-oxo-3,8-dioxa-5,11-diazatridecan-13-oicacid

Compound 43E (1 g, 2.37 mmol, 1.00 equiv) was dissolved in 40 mL of MeOHin the presence of Pd/C (1 g) and hydrogenated for 1 hour at ambienttemperature and atmospheric pressure. The reaction medium was filteredand concentrated under reduced pressure to yield 600 mg (76%) ofcompound 43F in the form of a white solid.

Compound 43G: tert-butyl((3R,4S,7S,10R)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2,14-dioxa-5,8,11-triazahexadecan-16-yl)(methyl)carbamate

Compound 43G was synthesised in the same manner as for compound 3 fromthe amine 1Y (50 mg, 0.08 mmol, 1.00 equiv), the acid 43F (50 mg, 0.08mmol, 1.00 equiv), DEPC (24.79 mg, 0.15 mmol, 2.00 equiv) and DIEA(29.46 mg, 0.23 mmol, 3.00 equiv) in DCM (1 mL). The crude product (59mg) was subsequently used as such.

Compound 43: Compound 43 was synthesised in the same manner as forcompound 2 from the intermediate 43G (81 mg, 0.08 mmol, 1.00 equiv). Thecrude product was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU,SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase: water/ACNbuffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220nm). Compound 43 was obtained with a yield of 64% (52.6 mg) in the formof a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeCN in water (0.05% TFA) for 8 minutes then 95% MeCNin water for 2 minutes); ESI (C₄₈H₇₇N₇O₇S, exact mass 871.6) m/z: 872.5(MH⁺⁾and 436.9 (M.2H⁺/2, 100%), 3.90 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.8-7.7 (m, 1H),7.55-4.45 (m, 1H), 7.35-7.1 (m, 5H), 5.5-5.75 (m, 1H), 4.9-4.6 (m, 2H),4.2-0.8 (m, 64H).

Compound 44 methyl(S)-2-((2R,3R)-3-((S)-1-((9S,12S,15S,16R)-15-((S)-sec-butyl)-9,12-diisopropyl-16-methoxy-8,14-dimethyl-10,13-dioxo-5-oxa-2,8,11,14-tetraazaoctadecan-18-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,bis trifluoroacetic acid

Compound 44A: methyl(S)-2-((2R,3R)-3-((S)-1-((12S,15S,18S,19R)-18-((S)-sec-butyl)-12,15-diisopropyl-19-methoxy-2,2,5,11,17-pentamethyl-4,13,16-trioxo-3,8-dioxa-5,11,14,17-tetraazahenicosan-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 44A was synthesised in the same manner as for compound 3 fromthe amine 3D (60 mg, 0.09 mmol, 1.00 equiv), the acid 43F (47 mg, 0.14mmol, 1.50 equiv), DEPC (31 mg, 0.19 mmol, 2.00 equiv) and DIEA (37 mg,0.28 mmol, 3.00 equiv) in DCM (1.5 mL). The crude product (58 mg) wassubsequently used as such.

Compound 44: Compound 44 was synthesised in the same manner as forcompound 2 from the intermediate 44A (58 mg, 0.06 mmol, 1.00 equiv). Thecrude product was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU,SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase: water/ACNbuffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10 minutes then40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220nm). Compound 44 was obtained with a yield of 40% (23.7 mg) in the formof a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeCN in water (0.05% TFA) for 8 minutes then 95% MeCNin water for 2 minutes); ESI (C₄₅H₇₈N₆O₉, exact mass 846.6) m/z: 847.6(MH⁺⁾and 424.4 (M.2H⁺/2, 100%), 3.20 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.3-7.1 (m, 5H),4.9-4.6 (m, 3H), 4.2-0.8 (m, 67H).

Compound 45(S)—N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(piperazin-1-yl)ethyl)amino)butanamido)butanamide,tris trifluoroacetic acid

Compound 45A: tert-butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate

2-(piperazin-1-yl)ethan-1-ol (5 g, 38.41 mmol, 1.00 equiv) was dissolvedin DCM (100 mL), and a solution of di-tert-butyl dicarbonate (8.38 g,38.40 mmol, 1.00 equiv) in DCM (20 mL) was added drop-wise. The reactionwas left under agitation overnight at ambient temperature. The reactionwas evaporated to dryness and the residue dissolved in 200 mL of AcOEt,washed 5 times with NaCl (sat.), dried over sodium sulfate, filtered andconcentrated under reduced pressure to yield 8.5 g (96%) of compound 45Ain the form of a white solid.

Compound 45B: tert-butyl 4-(2-oxoethyl)piperazine-1-carboxylate

Compound 45B was prepared in the same manner as for compound 35B, fromcompound 45A (1 g, 4.34 mmol, 1.00 equiv), oxalyl chloride (610 mg, 4.80mmol, 1.12 equiv), TEA (2.13 g, 21.09 mmol, 4.90 equiv) and DMSO (0.82g, 2.40 equiv). Compound 45B (0.8 g, 81%) was isolated in the form of acolourless oil.

Compound 45C: tert-butyl(S)-4-(2-((1-(benzyloxy)-3-methyl-1-oxobutan-2-yl)(methyl)amino)ethyl)piperazine-1-carboxylate

Compound 45C was synthesised in the same manner as for compound 14C fromthe amine 1ZC (720 mg, 2.79 mmol, 0.80 equiv), the aldehyde 45B (800 mg,3.50 mmol, 1.00 equiv), NaBH(OAc)₃ (1.6 g, 7.55 mmol, 2.15 equiv) andDIEA (2.5 mL) in THF (50 mL). The reaction mixture was neutralised with5 mL of water and extracted 3 times with 5 mL of AcOEt. The organicphases were combined, dried over sodium sulfate, filtered andconcentrated. The residue was purified on silica gel (EtOAc/PE (3:1) toyield 400 mg (33%) of compound 45C in the form of a colourless oil.

Compound 45D: (S)-2-((2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethyl)(methyl)amino)-3-methylbutanoic acid

Compound 45C (400 mg, 0.92 mmol, 1.00 equiv) was dissolved in 30 mL ofMeOH in the presence of Pd/C (400 mg) and hydrogenated for 1 hour atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure to yield 300 mg (95%)of compound 45D in the form of a white solid.

Compound 45E: tert-butyl4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)piperazine-1-carboxylate

Compound 45E was synthesised in the same manner as for compound 3 fromthe amine 1Y (60 mg, 0.09 mmol, 1.00 equiv), the acid 45D (62.7 mg, 0.18mmol, 2.00 equiv), DEPC (0.0278 mL) and DIEA (0.0452 mL) in DCM (3 mL).The crude product (100 mg) was subsequently used as such.

Compound 45: Compound 45 was synthesised in the same manner as forcompound 2 from the intermediate 45E (100 mg, 0.10 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254nm and 220 nm). Compound 45 was obtained with a yield of 19% (19.4 mg)in the form of a white solid.

LC/MS/UV (Agilent ZORBAX SB-Aq column, 1.8 μm, 4.6×100 mm; 40° C.; 1.0mL/min, 2% MeOH in water (0.05% TFA) for 1 minute then 2% to 95% MeOH inwater in 13 minutes then 95% MeOH in water for 2 minutes); ESI(C₄₇H₇₈N₈O₆S, exact mass 882.6) m/z: 883.5 (MH⁺) and 442.4 (M.2H⁺/2,100%), 10.95 min (98.8%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers), 7.80-7.70 (m,1H), 7.52-7.43 (m, 1H), 7.31-7.09 (m, 5H), 5.70-5.51 (m, 1H), 4.80-4.60(m, 1H), 4.20-0.75 (m, 66H).

Compound 46 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(piperazin-1-yl)ethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,tris trifluoroacetic acid

Compound 46A: tert-butyl4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)piperazine-1-carboxylate

Compound 46A was synthesised in the same manner as for compound 3 fromthe amine 3D (170 mg, 0.27 mmol, 1.00 equiv) the acid 45D (184.6 mg,0.54 mmol, 2.00 equiv), DEPC (0.0819 mL) and DIEA (0.133 mL) in DCM (5mL). The crude product (200 mg) was subsequently used as such.

Compound 46: Compound 46 was synthesised in the same manner as forcompound 2 from the intermediate 46A (100 mg, 0.10 mmol, 1.00 equiv).The crude product was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/ACN buffered with 0.05% TFA; Gradient of 20% to 40% ACN in 10minutes then 40% to 95% ACN in 2 minutes; Waters 2545 UV Detector at 254nm and 220 nm). Compound 46 was obtained with a yield of 19% (19.1 mg)in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeCN in water (0.05% TFA) for 8 minutes then 95% MeCNin water for 2 minutes); ESI (C₄₆H₇₉N₇O₈, exact mass 857.6) m/z: 858.6(MH⁺) an 429.9 (M.2H⁺/2, 100%), 5.93 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.58-8.50 (m,0.5H, NHCO, incomplete exchange), 8.29-8.22 (m, 0.4H, NHCO, incompleteexchange), 7.35-7.15 (m, 5H), 4.87-4.69 (m, 3H), 4.22-0.82 (m, 68H).

Compound 47(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(piperazin-1-yl)ethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, tris trifluoroacetic acid

Compound 47 was prepared in the same manner as for compound 4, fromcompound 46 (100 mg, 0.10 mmol, 1.00 equiv). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 32.6 mg(33%) of compound 47 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₆H₇₇N₇O₈, exact mass 843.6) m/z: 844.6 (M^(H)) and 422.9 (M.2H⁺/2,100%), 5.73 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.66-8.57 (m,0.3H, NHCO, incomplete exchange), 8.41-8.32 (m, 0.3H, NHCO, incompleteexchange), 8.13-8.06 (m, 0.2H, NHCO, incomplete exchange), 7.30-7.10 (m,5H), 4.80-4.61 (m, 3H), 4.19-0.78 (m, 65H).

Compound 48(S)-2-((S)-2-(((1H-imidazol-2-yl)methyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 48 was prepared in the same manner as for compound 1, from theamines 1Y and 1ZC and 1H-imidazole-2-carbaldehyde. The end product waspurified by preparative HPLC under the following conditions: SunFirePrep C18 OBD column, 5 μm, 19×150 mm, mobile phases buffered with 0.05%TFA, gradient of 15.0 to 30% ACN in water in 10 minutes then up to 95.0%ACN in 2 minutes, UV Detection UV 220 nm.

LC/MS/UV (Zorbax Eclipse Plus C8, 1.8 μm, 4.6×100 mm; 1 mL/min, 40° C.,2% methanol in water (eluting phases buffered with 0.05% TFA) for 1minute, then 2% to 95% methanol for 12 minutes; ESI (C₄₅H₇₀N₈O₆S, exactmass 850.51) m/z: 851.2 (MH⁺), 873.5 (MNa⁺), 426.3 (M.2H⁺/2); 12.75 min(90.5%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.83-7.81 (m,1H), 7.80-7.53 (m, 3H), 7.53-7.22 (m, 5H), 5.6-5.8 (m, 1H), 5.0-4.6 (m,2H); 4.6-0.85 (m, 55H).

Compound 49(S)-2-((S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide,trifluoroacetic acid

Compound 49A: 2-(4-hydroxyphenyl)acetaldehyde

4-(2-hydroxyethyl)phenol (4 g, 28.95 mmol, 1.00 quiv) was dissolved inDMSO (32 mL) and TEA (8.8 mL, 2.20 equiv) was then added dropwise. Asolution of SO₃.Py (10 g, 2.20 equiv) in DMSO (36 mL) was added and themixture was left under agitation overnight at ambient temperature. Thereaction mixture was neutralised with 250 mL of water and extracted 3times with 100 mL of AcOEt. The organic phases were combined, washed 5times with water (100 mL) then twice with 150 mL of NaCl (sat.), driedover sodium sulfate, filtered and concentrated. The residue was purifiedon silica gel (EtOAc/PE (1:10) to yield 1 g (25%) of compound 49A in theform of a colourless oil.

Compound 49B: benzyl(S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanoate

Compound 49B was synthesised in the same manner as for compound 14C fromthe amine 1ZC (1.5 g, 5.82 mmol, 0.99 equiv), the aldehyde 49A (800 mg,5.88 mmol, 1.00 equiv), NaBH(OAc)₃ (2.7 g, 12.74 mmol, 2.17 equiv) andDIEA (4.23 mL) in THF (25 mL). The reaction mixture was neutralised with50 mL of water and extracted 3 times with 50 mL of AcOEt. The organicphases were combined, dried over sodium sulfate, filtered andconcentrated. The residue was purified on silica gel (EtOAc/PE (1:10) toyield 600 mg (37%) of compound 49B in the form of a white solid.

Compound 49C: (S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanoicacid

Compound 49B (0.5 g, 1.46 mmol, 1.00 equiv) was dissolved in 40 mL ofMeOH in the presence of Pd/C (250 mg) and hydrogenated for 3 hours atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure to yield 0.4 g ofcompound 49C in the form of a white solid.

Compound 49: Compound 49 was synthesised in the same manner as forcompound 3 from the amine 1Y (53.4 mg, 0.08 mmol, 2.00 equiv), the acid49C (70 mg, 0.28 mmol, 1.00 equiv), DEPC (0.032 mL, 2.00 equiv) and DIEA(0.053 mL, 3.00 equiv) in DCM (3 mL). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 45% ACN in 10 minutes then 45% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 3 mg(1%) of compound 49 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₉H₇₄N₆O₇S, exact mass 890.5) m/z: 891.5 (MH⁺) and 446.4 (M.2H⁺/2,100%), 6.69 min (100%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.92-8.87 (m,0.5H, NHCO, incomplete exchange), 8.70-8.63 (m, 0.4H, NHCO, incompleteexchange), 8.85-8.77 (m, 1H), 7.59-7.51 (m, 1H), 7.35-7.03 (m, 7H),6.82-6.71 (m, 2H), 5.77-5.58 (m, 1H), 5.81-5.70 (m, 1H), 4.21-0.80 (m,58H).

Compound 50(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid, trifluoroacetic acid

Compound 50 was prepared in the same manner as for compound 4, fromcompound 27 (100 mg, 0.10 mmol, 1.00 equiv). The residue was purified bypreparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05% TFA;Gradient of 20% to 40% ACN in 10 minutes then 40% to 100% ACN in 2minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 10.7 mg(11%) of compound 50 in the form of a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6×100 mm; 40° C.; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) in 8 minutes); ESI(C₄₇H₇₃N₅O₉, exact mass 851.5) m/z: 852.5 (MH⁺) and 426.8 (M.2H⁺/2,100%), 6.46 min (91.7%, 210 nm).

¹H NMR: (400 MHz, CD₃OD, ppm): δ (Presence of rotamers) 7.34-7.15 (m,5H); 7.15-7.04 (se, 2H), 6.82-6.83 (m, 2H), 4.83-4.70 (m, 1H), 4.21-4.00(m, 1H), 3.90-3.80 (m, 1H), 3.74-3.62 (m, 1H), 3.57-2.86 (m, 20H),2.56-0.80 (m, 36H).

Compound 51 methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-hydroxybenzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate,trifluoroacetic acid

Compound 51A: tert-butyl (4-formylphenyl)carbonate

4-hydroxybenzaldehyde (3.0 g, 24 mmol) was dissolved in 30 mL of DCM inthe presence of 4-DMAP (300 mg, 2.46 mmol, 0.1 equiv.) and di-tert-butyldicarbonate (5.35 g, 24 mmol, 1.0 equiv.) and agitated 1 hour at ambienttemperature. The solution was then diluted with 200 mL of water andextracted 3 times with 100 mL of DCM. The organic phases were combined,dried over sodium sulfate, filtered and concentrated under reducedpressure to yield 5 g (92%) of compound 51A in the form of a whitesolid.

Compound 51B: benzyl (S)-2-((4-((tert-butoxycarbonyl)oxy)benzyl)(methyl)amino)-3-methylbutanoate

Compound 51A (220 mg, 0.99 mmol) was dissolved in 5 mL of THF in thepresence of compound 1ZC (255 mg, 0.99 mmol, 1.0 equiv.), NaBH(OAc)₃(420 mg, 2 mmol, 2.0 equiv.) and DIEA (654 μl) and agitated overnight atambient temperature. The solution was then diluted with 100 mL of waterand extracted 3 times with 50 mL of EtOAc. The organic phases werecombined, dried over sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified on a silica column with amixture of EtOAc and PE (1:100) to yield 200 mg (47%) of compound 51B inthe form of a white solid.

Compound 51C:(S)-2-((4-((tert-butoxycarbonyl)oxy)benzyl)(methyl)amino)-3-methylbutanoic acid

Compound 51C was prepared by hydrogenation of compound 51B (200 mg),following the protocol used for the preparation of compound 3F.

Compound 51D: methyl(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((tert-butoxycarbonyl)oxy)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

Compound 51D was prepared by coupling compound 51C with amine 3D,following the protocol used for the preparation of compound 3 to obtainthe desired product in the form of yellow oil with a yield of 60%.

Compound 51: Compound 51D (80 mg, 0.08 mmol) was dissolved in 1 mL ofDCM in the presence of 0.5 mL TFA, agitated 2 hours at ambienttemperature and then concentrated under reduced pressure. The residuewas purified by preparative HPLC (Pre-HPLC-010, SunFire Prep C18 OBDcolumn, 5 μm, 19×150 mm; Eluting phase: water/ACN buffered with 0.05%TFA; Gradient of 23% to 40% ACN in 10 minutes then 40% to 95% ACN in 2minutes; Waters 2489 UV Detector at 254 nm and 220 nm). Compound 51 wasobtained with a yield of 24% (20 mg) in the form of a white solid.

LC/MS/UV (Zorbax SB-Aq, 1.8 μm, 4.6×100 mm; 2% MeOH in water (0.05% TFA)for 1 minute then 2% to 95% MeOH in 13 minutes); ESI (C₄₇H₇₃N₅O₉, exactmass 851.54) m/z: 874.5 (MNa⁺), 426.9 (M.2H⁺/2); 12.48 min (96%, 210nm).

¹H NMR: (300 MHz, CD₃OD, ppm): δ (Presence of rotamers) 8.1-8.6 (m,0.9H, NHCO incomplete exchange); 7.29-7.27 (m, 2H), 7.25-6.86 (m, 5H),6.84-6.83 (m, 2H), 4.83-4.72 (m, 3H), 4.26-0.82 (m, 58H).

Compound 61(S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide

Compound 61A: N-(4-aminophenethyl)-N-methyl-L-valine dihydrochloride

Compound 11D (962 mg, 2.75 mmol) was dissolved in 10 ml of acommercially available solution of HCl in propan-2-ol (5-6 M), andstirred at room temperature for 2 hours. TLC analysis indicated completeconsumption of starting material. The solvent was evaporated underreduced pressure, and the resulting yellow solid triturated with Et₂O(2×10 ml). The product was dried under vacuum to furnish compound 61A asa yellow solid (322 mg, 47%).

Compound 61: Carboxylic acid 61A (73 mg, 0.23 mmol, 1 eq.) and amine 1Y(150 mg, 0.23 mmol, 1 eq.) were dissolved in dry DMF (2 ml). DIEA (158μl, 0.90 mmol, 4 eq.) and DECP (51 μl, 0.34 mmol, 1.5 eq.) were addedand the reaction stirred for 4 hours at room temperature. Analysis byLC-MS showed complete consumption of the starting material. The solventwas evaporated under reduced pressure, and the residue purified by flashchromatography on silica gel (DCM/MeOH) to furnish compound 61 as alight yellow solid (83 mg, 40%).

¹H NMR: (500 MHz, DMSO-d₆, ppm): δ (Presence of rotamers), 8.86 (d,0.5H, NHCO); 8.65 (d, 0.5H, NHCO), 8.11-8.05 (m, 1H, NHCO), 7.80 (d,0.5H, thiazole), 7.78 (d, 0.5H, thiazole), 7.65 (d, 0.5H, thiazole),7.63 (d, 0.5H, thiazole), 7.32-7.12 (m, 5H), 6.83 (d, J=8.3 Hz, 2H),6.45 (d, J=8.3 Hz, 2H), 5.56-5.49 (m, 0.5H), 5.42-5.35 (m, 0.5H), 4.78(s, 2H, NH₂), 4.74-4.46 (m, 2H), 4.01-0.66 (m, 57H).

HPLC (Xbridge Shield C18, 3.5 μm, 4.6×50 mm; 3.5 ml/min, 40° C., 0 to95% MeCN in water (0.1% TFA) in 2.25 minutes then 95% MeCN for 0.5minutes, Tr=1.31 min (96.5%, 220 nm).

m/z (Q-TOF ESI⁺) 890.5558 (2%, MH⁺, C₄₉H₇₆N₇O₆S requires 890.5572),445.7834 (100%, (MH₂)²⁺, C₄₉H₇₇N₇O₆S requires 445.7823).

Compound 62 Methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

Compound 62 was prepared in the same manner as for compound 61, usingcarboxylic acid 61A (69 mg, 0.21 mmol, 1 eq.), amine 3D (135 mg, 0.21mmol, 1 eq.), DIEA (75 μl, 0.43 mmol, 2 eq.) and DECP (49 μl, 0.32 mmol,1.5 eq.). The crude product was purified by flash chromatography onsilica gel (DCM/MeOH) to furnish compound 62 as a yellowish solid (82mg, 45%).

¹H NMR: (500 MHz, DMSO-d₆, ppm): δ (Presence of rotamers), 8.50 (d,J=8.3, 0.5H, NHCO); 8.27 (d, J=8.0, 0.5H, NHCO), 8.15-8.04 (m, 1H,NHCO), 7.27-7.13 (m, 5H), 6.86-6.79 (m, 2H), 6.48-6.42 (m, 2H), 4.78 (s,2H, NH₂), 4.74-4.44 (m, 3H), 4.01-3.72 (m, 1.5H), 3.66 (s, 1.5H, CO₂Me),3.63 (s, 1.5H, CO₂Me), 3.57-0.65 (m, 55.5H).

HPLC (Xbridge Shield C18, 3.5 μm, 4.6×50 mm; 3.5 ml/min, 40° C., 0 to95% MeCN in water (0.1% TFA) in 2.25 minutes then 95% MeCN for 0.5minutes, Tr=1.29 min (95.3%, 220 nm). m/z (Q-TOF ESI⁺) 865.5800 (2%,MH⁺, C₄₈H₇₇N₆O₈ requires 865.5797), 433.2937 (100%, (MH₂)²⁺, C₄₈H₇₈N₆O₈requires 433.2935).

Compound 63((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound 62 (23 mg, 0.03 mmol) was dissolved in a mixture of water (1ml) and acetonitrile (1 ml). Piperidine (0.75 ml) was added and themixture stirred at room temperature for 5 hours. TLC analysis indicatedcomplete consumption of the starting material. The solvent wasevaporated under reduced pressure, and the residue purified bypreparative HPLC (SunFire Prep column C18 OBD, 5 μm, 19×150 mm; Mobilephase: water/MeCN buffered with 0.1% TFA; Gradient of 20% to 40% MeCN in10 minutes, then from 40% to 100% MeCN in 2 minutes; Detector UV Waters2545 at 254 nm et 220 nm). Compound 63 was obtained as a white solid (14mg, 66%).

¹H NMR: (500 MHz, DMSO-d₆, ppm): δ (Presence of rotamers), 12.7 (s(br),1H, CO₂H), 9.58 (m(br), 1H); 9.04-8.89 (m, 1H), 8.41 (d, 0.6H, NHCO),8.15 (d, 0.4H, NHCO), 7.27-7.13 (m, 5H), 7.13-6.99 (m(br), 2H),6.90-6.64 (s(br), 2H), 4.77-3.40 (m, 10H), 3.34-2.75 (m, 20H), 2.34-1.94(m, 4H), 1.90-0.7 (m, 25H).

HPLC (Xbridge Shield C18, 3.5 μm, 4.6×50 mm; 3.5 ml/min, 40° C., 0 to95% MeCN in water (0.1% TFA) in 2.25 minutes then 95% MeCN for 0.5minutes, Tr=1.24 min (100%, 220 nm).

m/z (Q-TOF ESI⁺) 851.5641 (6%, MH⁺, C₄₇H₇₅N₆O₈ requires 851.5641),426.2854 (100%, (MH₂)²⁺, C₄₇H₇₆N₆O₈ requires 426.2857).

Example 20 Antiproliferative Activity of the Drugs

Method:

Cell culture. A549 (Non Small Cell Lung Cancer—ATCC CCL-185) andMDA-MB-231 (breast adenocarcinoma—ATCC HTB-26) cells were cultured inMinimum Essential Medium Eagle (MEM) with 5% fetal calf serum (FCS) andDulbecco's modified Eagle Medium (DMEM) with 10% FCS respectively. MCF7(breast ductal carcinoma—ATCC HTB-22) and SN-12C (kidney carcinoma—ATCC)cells were maintained in RPMI1640 medium (without phenol red for MCF7cells) containing 10% FCS. All the media were supplemented withfungizone (1.25 μg/mL) and penicillin-streptomycin (100 U/100 μg/mL).Cells were cultured under standard conditions in an incubator at 37° C.,5% CO₂ and 95% atmospheric humidity.

Antiproliferative activity on 4 tumor cell lines. Selected drugs wereinvestigated for their antiproliferative activity using an ATPliteproliferation assay (Perkin Elmer, Villebon sur Yvette, France) on acomprehensive panel of 4 cell lines. Cells were seeded in 96 well plates(10³ cells/well for A549, 2·10³ for MCF7, MDA-MB-231 and SN12C) at day 0at a concentration to ensure cells remained in logarithmic cell growthphase throughout the 72 h drug treatment period. After a 24 h incubationperiod, all the cells were treated with serial dilutions of the testedcompounds (11 μL of a 10× solution in 1% DMSO—6 wells/condition). Toavoid adherence of the compounds onto the tips, tips were changedbetween two consecutive dilutions. Cells were then placed in 37° C., 5%CO₂ incubator. On day 4, cell viability was evaluated by dosing the ATPreleased by viable cells. The number of viable cells was analyzed incomparison with the number of solvent treated cells. The EC₅₀ valueswere determined with curve fitting analysis (non linear regression modelwith a sigmoidal dose response, variable hill slope coefficient),performed with the algorithm provided by the GraphPad Software (GraphPadSoftware Inc., CA, USA).

Results:

Various Drugs:

Various drugs were tested to determine their antiproliferative activityon the MDA-MB-231 cell line following the above-described method. Themeasured activities gave values of EC₅₀<0.1 μM.

The few following examples chosen from among the above exemplified drugsillustrate their fully remarkable antiproliferative properties:

Example 12: EC₅₀=5.80×10⁻¹⁰ M; Example 13: EC₅₀=7.95×10⁻⁸ M; Example 15:EC₅₀=1.70×10⁻¹⁰ M; Example 27: EC₅₀=1.20×10⁻¹⁰ M.

Various Cell Lines:

Compound 15 was tested on different cell lines (A549, MDA-MB-231, MCF-7,SN12C) following the above-described method. The measured activitiesgave values of EC₅₀<0.1 μM on all the tested cell lines.

EC₅₀ (M) A549 MDA-MB-231 MCF-7 SN12C Compound 15 1.45 × 10⁻¹⁰ 1.70 ×10⁻¹⁰ 7.15 × 10⁻¹⁰ 2.18 × 10⁻¹⁰

Comparative examples:

The substitution on the phenyl ring (amino/hydroxyl v. carboxyl) wasstudied in the comparative examples below showing the improvedantiproliferative activity of the drugs according to the inventioncomprising an amino or hydroxyl substituent.

EC₅₀ (M) MDA- No Structure A549 MB-231 12

1.48 × 10⁻¹⁰ 5.80 × 10⁻¹⁰ 15

1.45 × 10⁻¹⁰ 1.70 × 10⁻¹⁰ 27

8.60 × 10⁻¹¹ 1.20 × 10⁻¹⁰ Com- para- tive exam- ple

3.76 × 10⁻⁹  2.29 × 10⁻⁹  13

2.71 × 10⁻⁸  7.95 × 10⁻⁸  Com- pra- tive exam- ple

4.03 × 10⁻⁷  9.75 × 10⁻⁷ 

Example 21 Synthesis of the Drug-Linker Moiety Compound E-114-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)phenyl)(methyl)carbamate2,2,2-trifluoroacetate

Compound E-11-1: methyl (S)-2-amino-5-ureidopentanoate hydrochloride

Acetyl chloride (10 mL) was added dropwise to MeOH (120 mL) at 0° C.with stirring. After 20 minutes, L-Citrulline (10 g, 57 mmol, 1.00 eq.)was added and the mixture heated at reflux overnight. The solvent wasevaporated under reduced pressure to yield 15 g (116%) of compoundE-11-1 as a white solid. The product was used in the next step withoutfurther drying.

Compound E-11-2: methyl(S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoate

Compound E-11-1 (13 g, 57.6 mmol, 1.1 eq.) was dissolved in DMF (140 mL)at 0° C. under an inert atmosphere. DIEA (30 mL, 173 mmol, 3.0 eq.),hydroxybenzotriazole (HOBt—10.59 g, 69.1 mmol, 1.2 eq.) and Boc-L-valinehydroxysuccinimide ester (Boc-Val-OSu—18.1 g, 57.6 mmol, 1.0 eq.) wereadded. The reaction mixture was agitated overnight at ambienttemperature, then the solvent was evaporated under reduced pressure. Theresidue was dissolved in water (100 mL) and extracted twice with DCM(150 mL). The organic phases were combined, dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified on silicagel (DCM/MeOH) to yield 18.8 g (84%) of compound E-11-2 as a whitesolid.

Compound E-11-3: (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid

Compound E-11-2 (18.8 g, 48.4 mmol, 1 eq.) was dissolved in MeOH (200mL) at 0° C. A solution of NaOH 1M (72 mL, 72 mmol, 1.5 eq.) was addedand the mixture stirred for 2 hours at room temperature. The MeOH wasremoved under reduced pressure and the remaining aqueous solutionacidified with HCl 1M. The aqueous phase was evaporated to dryness andthe residue purified on silica gel (DCM/MeOH) to yield 18 g (99%) ofcompound E-11-3 as a white solid.

Compound E-11-4: tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

Compound E-11-3 (5 g, 13.4 mmol, 1 eq.) was dissolved in a mixture ofdry DCM (65 ml) and dry MeOH (35 ml). (4-aminophenyl)methanol (1.81 g,14.7 mmol, 1.1 eq.) and N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ—6.60 g, 26.7 mmol, 2 eq.) were added and the mixture stirred inthe dark overnight. The solvents were evaporated under reduced pressure,and the residue purified on silica gel (DCM/MeOH) to yield 5.2 g (73%)of compound E-11-4 as an off-white solid.

Compound E-11-5: tert-butyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate

Compound E-11-4 (1.1 g, 2.29 mmol, 1 eq.) was dissolved in dry DMF (5ml) at ambient temperature under an inert atmosphere. Bis(4-nitrophenyl)carbonate (1.40 g, 4.59 mmol, 2 eq.) was added, followed by DIEA (600μl, 3.44 mmol, 1.5 eq.), and the resulting yellow solution stirredovernight. The DMF was evaporated under reduced pressure, and theresidue purified on silica gel (DCM/MeOH) to yield 1.27 g (84%) ofcompound E-11-5 as an off-white solid.

Compound E-11-6:4—((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)phenyl)(methyl)carbamate2,2,2-trifluoroacetate

Carbonate E-11-5 (114 mg, 0.177 mmol, 1.2 eq.) and aniline 11F (150 mg,0.147 mmol, 1 eq.) were dissolved in dry DMF (4 mL). HOBt (38 mg, 0.295mmol, 2 eq.) and DIEA (54 μL, 0.295 mmol, 2 eq.) were added and themixture stirred for the weekend at room temperature. The DMF wasevaporated under reduced pressure and the residue purified by flashchromatography on silica, eluting with DCM. The product was repurifiedby preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 μm,19×100 mm; Eluting phase: water/MeCN buffered with 0.1% TFA; Gradient of5% to 100% MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). Theselected fractions were combined and lyophilised to furnish compoundE-11-6 as a white solid (89 mg, 39%).

Compound E-11

Compound E-11-6 (21 mg, 0.014 mmol, 1.0 eq.) was dissolved in DCM (0.25mL) and TFA (40 μL) was added. The solution was stirred for 2 hours atroom temperature, after which, LC-MS analysis indicated completeconsumption of starting material. The mixture was briefly cooled (bathof liquid nitrogen) whilst simultaneously adding DMF (0.5 mL) then DIEA(100 μL) in order to neutralise the TFA. The cooling bath was thenremoved and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (4 mg, 0.012 mmol, 1eq.) was added. The mixture was stirred at room temperature for 48 hoursand the product purified by preparative HPLC (Waters 600E, SunFire PrepC18 OBD column, 5 μm, 19×100 mm; Eluting phase: water/MeCN buffered with0.1% TFA; Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UVDetector at 220 nm). The selected fractions were combined andlyophilised to furnish compound E-11 as a white solid (11 mg, 54%).

m/z (Q-TOF MS ESI+) 1524.8282 (2%, MNa⁺, C₇₉H₁₁₅N₁₃NaO₁₄S requires1524.8299), 751.9283 (100%, (MH₂)²⁺, C₇₉H₁₁₇N₁₃O₁₄S requires 751.9276).

Compound E-12 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound E-12-1: tert-butyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((perfluorophenoxy)carbonyl)oxy)methyl)phenyl)amino)-5-ureidopentan-2-yl)amino)butan-2-yl)carbamate

Compound E-11-4 (670 mg, 1.26 mmol, 1 eq.) was dissolved in dry DMF (6ml) at 0° C. under an inert atmosphere. Bis(perfluorophenyl) carbonate(991 mg, 2.51 mmol, 2 eq.) was added, followed by DIEA (329 μl, 1.89mmol, 1.5 eq.), and the resulting colourless solution stirred for 30minutes at room temperature. The DMF was evaporated under reducedpressure, and the residue purified on silica gel (DCM/MeOH) to yield 836mg (96%) of compound E-12-1 as an off-white solid.

Compound E-12-2: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Aniline 12 (165 mg, 0.189 mmol, 1.0 eq.) was dissolved in DMF (5 mL) at0° C. under an inert atmosphere. Carbonate E-12-1 (194 mg, 0.282 mmol,1.5 eq.), HOBt (51 mg, 0.375 mmol, 2 eq.) and DIEA (66 μL, 0.375 mmol, 2eq.) were added and the mixture stirred at room temperature for 8 hours.The solvent was evaporated under reduced pressure and the residuepurified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column,5 μm, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1% TFA;Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UV Detector at220 nm). The selected fractions were combined and lyophilised to furnishcompound E12-7 as a white solid (247 mg, 77%).

Compound E-12-3: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninatebis(2,2,2-trifluoroacetate)

Compound E-12-2 (5.6 mg, 4.04 μmol, 1.0 eq.) was dissolved TFA (100 μL).After 5 minutes, 2 ml of water was added and the mixture lyophilisedovernight to yield compound E-12-3 as an off-white solid (5.6 mg, 98%).

Compound E-12

Compound E-12-3 (5.6 mg, 4 μmol, 1.0 eq.) was dissolved in acetonitrile(0.5 mL), and DIEA (5 μL, 7 eq.) was added, followed by2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.5 mg, 8 μmol, 2eq.). The mixture was stirred for 6 hours at room temperature. Aftercontrolling the reaction by LC-MS, 200 μL of water was added, and theresulting solution purified by preparative HPLC (Waters 600E, SunFirePrep C18 OBD column, 5 μm, 19×100 mm; Eluting phase: water/MeCN bufferedwith 0.1% TFA; Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UVDetector at 220 nm). The selected fractions were combined andlyophilised to furnish compound E-12 as a white solid (4.6 mg, 70%).

m/z (Q-TOF MS ESI+) 739.4389 (100%, (MH₂)²⁺, C₇₈H₁₁₈N₁₂O₁₆ requires739.4389).

Compound E-13((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound E-13-1:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

Compound E-12-2 (185 mg, 0.123 mmol, 1.0 eq.) was dissolved in a mixtureof water (5 mL) and acetonitrile (5 mL) at room temperature. Piperidine(3.67 mL, 300 eq.) was added and the mixture stirred for 6 hours at roomtemperature. The solvents were evaporated to dryness under reducedpressure, and the residue triturated with Et₂O (60 mL). The solid wasrinsed with twice Et₂O (20 ml) and dried under vacuum to yield compoundE-13-1 as an off-white solid (175 mg, 95%).

Compound E-13-2:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninebis (2,2,2-trifluoroacetate)

Compound E-13-1 (175 mg, 0.128 mmol, 1.0 eq.) was dissolved TFA (200μL). After 5 minutes, water (1 mL) and acetonitrile (1 mL) were addedand the solution lyophilised overnight to yield compound E-13-2 as anoff-white solid (180 mg, 87%).

Compound E-13:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine,2,2,2-trifluoroacetate

Compound E-13-2 (80 mg, 0.058 mmol, 1.0 eq.) as dissolved in a mixtureof acetonitrile (1.5 mL) and DMF (0.4 mL). DIEA (50 μL, 0.289 mmol, 5eq.) was added, followed by 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (36 mg, 0.116 mmol, 2eq.). The mixture was stirred for 3 hours at room temperature. Aftercontrolling the reaction by LC-MS, the solvent was evaporated underreduced pressure and the residue purified by preparative HPLC (Waters600E, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/MeCN buffered with 0.1% TFA; Gradient of 5% to 100% MeCN in 15minutes; Waters 2487 UV Detector at 220 nm). The selected fractions werecombined and lyophilised to furnish compound E-13 as a white solid (32mg, 35%).

mz (Q-TOF MS ESI−) 1461.8336 (100%, (M-H)⁻, C₇₇H₁₁₃N₁₂O₁₆ requires1461.8403).

mz (Q-TOF MS ESI+) 1463.8565 (2%, MH⁺, C₇₇H₁₁₅N₁₂O₁₆ requires1463.8549), 732.4317 (100%, (MH₂)²⁺, C₇₇H₁₁₆N₁₂O₁₆ requires 732.4311).

Compound E-15 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound E-15-1: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound E-15-1 was prepared according to the same method as forcompound E-11-6, using carbonate E-11-5 (28 mg, 0.044 mmol, 1 eq.),aniline 15 (42 mg, 0.044 mmol, 1 eq.), HOBt (3 mg, 0.022 mmol, 0.5 eq.),and DIEA (15 μL, 0.087 mmol, 2 eq.) in DMF (2 mL). Compound E-15-1 wasisolated as a white solid (8.2 mg, 13%).

Compound E-15-2: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninatebis(2,2,2-trifluoroacetate)

Compound E-15-1 (8.2 mg, 5.58 μmol, 1.0 eq.) was dissolved in TFA (200μL). After 5 minutes, water (1 mL) was added and the solutionlyophilised overnight to yield compound E-15-8 as a white solid (7.6 mg,99%).

Compound E-15

Compound E-15 was prepared according to the same method as for compoundE-12, using amine E-15-2 (7.6 mg, 5.55 μmol, 1 eq.),2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2 mg, 6.65 μmol, 1.2eq.) and DIEA (5 μL, 0.028 mmol, 5 eq.) in acetonitrile (0.5 mL).Compound E-15 was isolated as a white solid (4.2 mg, 48%). m/z (Q-TOF MSESI+) 1471.8169 (2%, MNa⁺, C₇₆H₁₁₂N₁₂NaO₁₆ requires 1471.8211), 725.4223(100%, (MH₂)²⁺, C₇₆H₁₁₄N₁₂O₁₆ requires 725.4232), 483.9482 (10%,(MH₃)³⁺, C₇₆H₁₁₅N₁₂O₁₆ requires 483.9513).

Compound F-13((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-N-methyl-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound F-13-1: benzyl N-(4-((tert-butoxycarbonyl)(methyl)amino)phenethyl)-N-methyl-L-valinate

Compound 11C (250 mg, 0.567 mmol, 1 eq.) was dissolved in THF (10 ml)followed by the addition of NaH (60% suspension in mineral oil, 68 mg,1.702 mmol, 3 eq.). The mixture was stirred for 5 minutes before addingiodomethane (106 μL, 1.702 mmol, 3 eq.). The reaction was stirred for 2hours at room temperature before quenching with water and separatingbetween EtOAc (100 mL) and water (50 mL). The organic phase was driedover MgSO₄ and evaporated to dryness to yield compound F-13-1 as ayellow oil (250 mg, 97%), which was used without further purification.

Compound F-13-2: benzyl N-methyl-N-(4-(methylamino)phenethyl)-L-valinate

Boc-protected aniline F-13-1 (250 mg, 0.550 mmol, 1 eq) was dissolved inMeOH (5 mL) followed by the addition of 1 mL of a commercially-availablesolution of HCl in ^(i)PrOH (5-6 M). The solution was stirred at roomtemperature for 2 hours before evaporating to dryness under reducedpressure. The resulting yellow oil was triturated with Et₂O to yieldcompound F-13-2 as a yellow solid (202 mg, 94%).

Compound F-13-3: benzylN-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-N-methyl-5-ureidopentanamido)phenethyl)-N-methyl-L-valinate

Acid E-11-3 (190 mg, 0.508 mmol, 1.5 eq.) was dissolved in dry DMF (1ml), followed by the addition of DIEA (118 μL, 0.677 mmol, 2 eq.),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP—264 mg, 0.508 mmol, 1.5 eq.) and aniline F-13-2 (120 mg, 0.339mmol, 1 eq.). The mixture was stirred at room temperature overnight andthe solvents evaporated under reduced pressure. The residue was purifiedby preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 μm,19×100 mm; Eluting phase: water/MeCN buffered with 0.1% TFA; Gradient of5% to 100% MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). Theselected fractions were combined and lyophilised to furnish compoundF-13-3 as a white solid (140 mg, 45%).

Compound F-13-4:N-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-N-methyl-5-ureidopentanamido)phenethyl)-N-methyl-L-valine

Compound F-13-3 (116 mg, 0.163 mmol, 1 eq.) was dissolved in MeOH (5 ml)in the presence of Pd/C 10% (30 mg) and hydrogenated for 2 hours atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure to yield 110 mg (99%)of compound F-13-4 as a beige solid.

Compound F-13-5: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-N-methyl-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Amine 3D (89 mg, 0.140 mmol, 1 eq.) and acid F-13-4 (145 mg, 0.210 mmol,1.5 eq.) were dissolved in dry DMF (4 mL), and PyBOP (109 mg, 0.210mmol, 1.5 eq.) and DIEA (73 μL, 0.420 mmol, 3 eq.) were added. Themixture was stirred for 1 hour at room temperature and the solventevaporated. The residue was separated between EtOAc and water, and theorganic phase dried over MgSO₄, filtered and evaporated under reducedpressure. The crude product was purified by preparative HPLC (Waters600E, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/MeCN buffered with 0.1% TFA; Gradient of 5% to 100% MeCN in 15minutes; Waters 2487 UV Detector at 220 nm). The selected fractions werecombined and lyophilised to furnish compound F-13-5 as a white solid(140 mg, 73%).

Compound F-13-6:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-N-methyl-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound F-13-5 (140 mg, 0.104 mmol, 1 eq.) was dissolved in a mixtureof water (4 mL), acetonitrile (4 mL) and piperidine (2 mL) and stirredat room temperature for 4 hours. The solvent was evaporated underreduced pressure and the residue purified by preparative HPLC (Waters600E, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Eluting phase:water/MeCN buffered with 0.1% TFA; Gradient of 5% to 100% MeCN in 15minutes; Waters 2487 UV Detector at 220 nm). The selected fractions werecombined and lyophilised to furnish compound F-13-6 as a white solid(115 mg, 83%).

Compound F-13

Compound F-13 was prepared according to the same method as for compoundE-11, using Boc-protected amine F-13-6 (55 mg, 0.041 mmol, 1.0 eq.) inDCM (0.5 mL) and TFA (100 μL, 30 eq.), followed by dilution with DMF (1mL), quenching with (DIEA (320 μL, 45 eq) then reaction with2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (15 mg, 0.049 mmol,1.2 eq.). After purification by preparative HPLC and lyophilisation,compound F-13 was obtained as a white solid (14 mg, 24%).

m/z (Q-TOF MS ESI+) 1314.8067 (2%, MH⁺, C₆₉H₁₀₈N₁₁O₁₄ requires1314.8072), 657.9067 (100%, (MH₂)²⁺, C₆₉H₁₀₉N₁₁O₁₄ requires 657.9072).

Compound F-61N—((S)-1-(((S)-1-((4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide2,2,2-trifluoroacetate

Compound F-61-1: benzyl N-(4-aminophenethyl)-N-methyl-L-valinatedihydrochloride

Compound 11C (1.0 g, 2.27 mmol, 1 eq.) was dissolved in 8 mL of acommercially-available solution of HCl in ^(i)PrOH (5-6 M). The mixturewas stirred for 2 hours at room temperature before evaporating todryness under reduced pressure. The residue was triturated twice withEt₂O (30 mL) and dried under vacuum to yield compound F-61-1 as a whitesolid (916 mg, 98%).

Compound F-61-2: benzylN-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)-N-methyl-L-valinate

Acid E-11-3 (769 mg, 2.05 mmol, 1.5 eq.) was dissolved in dry DMF (2.5ml) followed by the addition of DIEA (957 μL, 5.48 mmol, 4 eq.) andPyBOP (1.07 g, 2.05 mmol, 1.5 eq.). Aniline F-61-1 (566 mg, 1.369 mmol,1 eq.) was added and the mixture stirred at room temperature overnight.The solvents were evaporated under reduced pressure, and the residuepurified on silica gel (DCM/MeOH) to yield 969 mg (102%) of compoundF-61-2 as a white solid.

Compound F-61-3:N-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)-N-methyl-L-valine

Compound F-61-2 (969 mg, 1.28 mmol, 1 eq.) was dissolved in MeOH (20 ml)in the presence of Pd/C 10% (270 mg) and hydrogenated for 3 hours atambient temperature and atmospheric pressure. The reaction medium wasfiltered and concentrated under reduced pressure, and the residuepurified on silica gel (DCM/MeOH/AcOH) to yield 520 mg (67%) of compoundF-61-3 as a white solid.

Compound F-61-4: tert-butyl((S)-1-(((S)-1-((4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate2,2,2-trifluoroacetate

Acid F-61-3 (67.5 mg, 0.111 mmol, 1.5 eq.) was dissolved in dry DMF (2mL) and DECP (17 μL, 0.111 mmol, 1.5 eq.) and DIEA (39 μL, 0.223 mmol, 3eq.) were added. After stirring for 15 minutes at room temperature,amine 1Y (50 mg, 0.074 mmol, 1 eq.) was added and the solution stirredovernight. The solvent was evaporated under reduced pressure, and theresidue purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBDcolumn, 5 μm, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1%TFA; Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UV Detectorat 220 nm). The selected fractions were combined and lyophilised tofurnish compound F61-4 as a white solid (28 mg, 28%).

Compound F-61-5:(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-7,10-diisopropyl-3-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazatridecan-13-yl)phenyl)-5-ureidopentanamidebis(2,2,2-trifluoroacetate)

Compound F-61-4 (28 mg, 0.021 mmol, 1.0 eq.) was dissolved in TFA (200μL). After 5 minutes, water (2 mL) and acetonitrile (0.5 mL) were addedand the solution lyophilised overnight to yield compound F-61-5 as acolourless oil (38 mg, 134%).

Compound F-61

Compound F-61-5 (28.3 mg, 0.020 mmol, 1 eq.) was dissolved inacetonitrile (0.5 mL), followed by 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (9 mg, 0.029 μmol, 1.4eq.) and DIEA (25 μL, 0.143 mmol, 7 eq.). The mixture was stirred for4.5 hours, after which time HPLC analysis showed the presence ofstarting material but complete consumption of the succinimide.Supplementary 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate was therefore added (3mg, 0.01 μmol, 0.5 eq.) and the reaction stirred for 1.5 hours. HPLCanalysis showed complete consumption of the starting material. Thesolvent was evaporated to dryness and the residue triturated twice witha mixture of EtOAc/Et₂O (80/20) to yield compound F-61 as an off-whitesolid (19.4 mg, 70%).

m/z (Q-TOF MS ESI+) 1361.7725 (2%, MNa⁺, C₇₀H₁₀₆N₁₂NaO₁₂S requires1361.7666), 670.3961 (100%, (MH₂)²⁺, C₇₀H₁₀₈N₁₂O₁₂S requires 670.3960).

Compound F-62 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound F-62-1: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound F-62-1 was prepared in similar manner to compound F-61-4 fromamine 3D (100 mg, 0.158 mmol, 0.9 eq.), acid F-61-3 (108 mg, 0.178 mmol,1 eq.), DECP (41 μL, 0.267 mmol, 1.5 eq.) and DIEA (93 μL, 0.534 mmol, 3eq.) in DMF (2 mL). After purification by preparative HPLC, compoundF-62-1 was obtained as a white solid (93 mg, 39%).

Compound F-62-2: methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninatebis(2,2,2-trifluoroacetate)

Compound F-62-1 (35 mg, 0.026 mmol, 1.0 eq.) was dissolved in TFA (200μL). After 10 minutes, water (2 mL) and acetonitrile (0.5 mL) were addedand the solution lyophilised overnight to yield compound F-62-2 as awhite solid (34 mg, 105%).

Compound F-62

Amine F-62-2 (34 mg, 5.55 mol, 1 eq.) was dissolved in acetonitrile (3mL). DIEA (5 μL, 0.028 mmol, 5 eq.) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2 mg, 6.65 mol, 1.2eq.) were added. HPLC analysis showed complete consumption of thestarting material. The solvent was evaporated to dryness and the residuetriturated with a mixture of EtOAc/Et₂O (80/20). The crude product waspurified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column,5 μm, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1% TFA;Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UV Detector at220 nm). The selected fractions were combined and lyophilised to furnishcompound F-62 as a white solid (5.5 mg, 13%).

m/z (Q-TOF MS ESI+) 1336.7859 (2%, MNa⁺, C₆₉H₁₀₇N₁₁NaO₁₄ requires1336.7891), 657.9073 (100%, (MH₂)²⁺, C₆₉H₁₀₉N₁₁O₁₄ requires 657.9072).

Compound F-63((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound F-63-1:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

Compound F-62-1 (157 mg, 0.118 mmol, 1 eq.) was dissolved in a mixtureof water (4.5 mL), acetonitrile (4.5 mL) and piperidine (3.5 mL) andstirred at room temperature for 5 hours. The solvent was evaporatedunder reduced pressure and the residue triturated Et₂O (60 mL). Thesolid was collected by filtration and rinsed twice with Et₂O (10 mL) toyield compound F-63-1 as an off-white solid (153 mg, 100%).

Compound F-63-2:((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine bis2,2,2-trifluoroacetate

Compound F-63-1 (153 mg, 0.127 mmol, 1.0 eq.) was dissolved in TFA (200μL). After 10 minutes, water (2 mL) and acetonitrile (0.5 mL) were addedand the solution lyophilised overnight to yield compound F-63-2 as awhite solid (34 mg, 105%).

Compound F-63

Amine F-63-2 (100 mg, 0.082 mmol, 1 eq.) was dissolved in a mixture ofacetonitrile (2 mL) and DMF (0.5 mL), and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (45 mg, 0.147 mmol,1.8 eq.) and DIEA (71 μL, 0.409 mmol, 5 eq.) were added. After stirringat room temperature for 4.5 hours, the solvent was evaporated underreduced pressure. The crude product was purified by preparative HPLC(Waters 600E, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Elutingphase: water/MeCN buffered with 0.1% TFA; Gradient of 5% to 100% MeCN in15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractionswere combined and lyophilised to furnish compound F-63 as a white solidafter (42 mg, 36%).

m/z (Q-TOF MS ESI+) 1300.7901 (2%, MH⁺, C₆₈H₁₀₆N₁₁O₁₄ requires1300.7915), 650.8990 (100%, (MH₂)²⁺, C₆₈H₁₀₇N₁₁O₁₄ requires 650.8994).

Compound G-12 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound G-12-1: benzyl N-(4-aminophenethyl)-N-methyl-L-valinatedihydrochloride

Into oxalyl chloride (3 mL) was dissolved6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (200 mg, 0.947mmol, 1 eq.). The solution was stirred at room temperature for 5 hoursbefore evaporating to dryness under reduced pressure. Compound G-12-1was obtained as a beige solid (217 mg, 100%) and used in the next stepwithout purification.

Compound G-12

Aniline 12 (40 mg, 0.045 mmol, 1 eq.) was dissolved in dry DCM (1 mL) at0° C. and DIEA (8 μL, 0.045 mmol, 1 eq.) was added. After stirring for30 minutes, a solution of compound G-12-1 (10 mg, 0.45 mmol, 1 eq.) indry DCM (1 mL) was introduced and the reaction stirred for 1 hour at 0°C. The mixture was diluted with DCM (25 ml) and washed twice with water(20 mL), once with brine (10 mL). The organic phase was dried overNa₂SO₄, filtered and evaporated under reduced pressure to yield thecrude product as a light brown solid (54 mg). This was purified by flashchromatography on silica gel (DCM/MeOH) followed by preparative HPLC(Waters 600E, SunFire Prep C18 OBD column, 5 μm, 19×100 mm; Elutingphase: water/MeCN buffered with 0.1% TFA; Gradient of 5% to 100% MeCN in15 minutes; Waters 2487 UV Detector at 220 nm). The isolated product waslyophilised to yield a white solid (23 mg), which was repurified bypreparative HPLC and the selected fractions combined and lyophilised tofurnish compound G-12 as a white solid (9 mg, 16%).

m/z (Q-TOF MS ESI+) 1094.6543 (20%, MNa⁺, C₅₉H₈₉N₇NaO₁₁ requires1094.6512), 1072.6722 (16%, MH⁺, C₅₉H₉₀N₇O₁₁ requires 1072.6693),536.8358 (100%, (MH₂)²⁺, C₅₉H₉₁N₇O₁₁ requires 536.8383).

Compound G-13((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine2,2,2-trifluoroacetate

Compound G-13

Aniline 13 (15 mg, 0.015 mmol, 1 eq.) was dissolved in dry DCM (1.5 mL)at 0° C. and DIEA (8 μL, 0.046 mmol, 3 eq.) was added. A solution ofcompound G-12-1 (3.5 mg, 0.046 mmol, 1 eq.) in dry DCM (0.5 mL) wasintroduced and the reaction stirred for 1.5 hours at 0° C. The solventwas evaporated under reduced pressure and the crude product purified bypreparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 μm, 19×100mm; Eluting phase: water/MeCN buffered with 0.1% TFA; Gradient of 5% to100% MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). Theselected fractions were combined and lyophilised to furnish compoundG-13 as a white solid (11.4 mg, 62%).

m/z (Q-TOF MS ESI+) 1058.6510 (30%, MH⁺, C₅₈H₈₈N₇O₁₁ requires1058.6536), 529.8285 (100%, (MH₂)²⁺, C₅₈H₈₉N₇O₁₁ requires 529.8305).

Compound G-15 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)benzyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate2,2,2-trifluoroacetate

Compound G-15

Aniline 15 (40 mg, 0.047 mmol, 1 eq.) was dissolved in dry DCM (2 mL) at0° C. and DIEA (10 μL, 0.056 mmol, 1.2 eq.) was added. A solution ofcompound G-12-1 (108 mg, 0.47 mmol, 10 eq.) in dry DCM (1 mL) wasintroduced and the reaction stirred for 1.5 hours at 0° C. The mixturewas diluted with DCM (10 ml) and washed twice with water (5 mL). Theorganic phase was dried over MgSO₄, filtered and evaporated underreduced pressure to yield the crude product as a beige solid. This waspurified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column,5 μm, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1% TFA;Gradient of 5% to 100% MeCN in 15 minutes; Waters 2487 UV Detector at220 nm). The selected fractions were combined and lyophilised to furnishcompound G15 as a white solid (27 mg, 50%).

m/z (Q-TOF MS ESI+) 1066.6517 (2%, MNa⁺, C₅₇H₈₅N₇NaO₁₁ requires1066.6199), 522.8224 (100%, (MH₂)²⁺, C₅₇H₈₇N₇O₁₁ requires 522.8226).

Example 22 ADC Synthesis, Purification and Characterization

The procedure described below applies to chimeric and humanized IgG1forms. It must be undertood that for any other forms, such as IgG2,IgG4, etc., the person skilled n the art would be capable of adaptingthis procedure using the general knowledge.

Antibodies (1-5 mg/ml) were partially reduced withTris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 10 mM boratebuffer pH 8.4 containing 150 mM NaCl and 2 mM EDTA for 2 h at 37° C.Typically, 2.5-3 molar equivalents of TCEP were used to target aDrug-to-Antibody Ratios (DAR) of around 4, respectively. The partialantibody reduction was confirmed by SDS-PAGE analysis under non reducingconditions. Before Linker-Drug coupling to the released interchaincysteine residues, the reduction mixture was allowed to cool to roomtemperature. The antibody concentration was then adjusted to 1 mg/mlwith 10 mM borate buffer pH 8.4 containing 150 mM NaCl and 2 mM EDTA,and a 5 molar excess of drug to antibody was added from a 10 mM solutionin dimethyl sulfoxide (DMSO). The final DMSO concentration was adjustedto 10% to maintain the solubility of the drug in the aqueous mediumduring coupling. The reaction was carried out for 1 h at roomtemperature. The drug excess was quenched by addition of 1.5 moles ofN-acetylcysteine per mole of drug and incubation for 1 h at roomtemperature. After dialysis against 25 mM His buffer pH 6.5 containing150 mM NaCl overnight at 4° C., the antibody-drug-conjugates werepurified by using methods known to persons skilled in the art based withcommercial chromatography columns and ultrafiltration units. First, thenon coupled drug and the ADC aggregates were eliminated by sizeexclusion chromatography (SEC) on S200 (GE Life Sciences) or TSK G3000SW (Tosoh) column. The purified ADC monomers were then concentrated to2-3 mg/ml by ultrafiltration on 30 or 50 kDa MWCO filtration units or byaffinity chromatography on Protein A. The purified ADCs were stored at4° C. after sterile filtration on 0.2 μm filter. They were furtheranalyzed by SDS-PAGE under reducing and non reducing conditions toconfirm drug conjugation and by SEC on analytical S200 or TSK G3000 SWXLcolumns to determine the content of monomers and aggregated forms.Protein concentrations were determined by using the bicinchoninic acid(BCA) assay with IgG as standard. The DAR was estimated for eachpurified ADC by HIC and LC-MS. Typically, the content of aggregatedforms was lower than 5% and the DAR was comprised between 3.5 and 5.

Example 23 Cytotoxicity Evaluation of IGF-1R Antibodies Coupled withDifferent Drugs

The five IGF-1R antibodies were shown to be rapidly internalized intolysosomes and to have a lower binding capacity into acidic environments.In that respect, those Abs had all properties to be used as ADCs. Thus,the five chimeric anti-IGF-1R antibodies were coupled with threedifferent compounds (G-13; E-13 and F-63). The drug antibody ratio ofthose ADCs was about 4. In order to evaluate the non specificcytotoxicity, an irrelevant chimeric antibody c9G4 was also coupled withthose compounds at the same DAR. MCF-7 cells were incubated withincreasing concentrations of each ADCs at 37° C. for 6 days in completeculture medium. Cell viability was assessed using a luminescent cellviability assay (CellTiter-Glo, Promega). Luminescent signal was readusing a the Mithras plate reader (Berthold Technologies). The irrelevantchimeric antibody c9G4 coupled with either E-13, G-13 or F-63 showed noor modest cytotoxic activity on MCF-7 cells (FIG. 21). On the contrary,addition of all other ADCs obtained after coupling anti-IGF-1Rantibodies with either E-13, G-13 or F-63 decreased dramatically MCF-7cell viability.

Example 24 In Vivo Activity of the c208F2 Antibody Conjugated to EitherE-13, G-13 or F-63 Compounds in the MCF-7 Xenograft Model

In order to confirm that the in vitro efficacy of the c208F2 coupled toG-13, E-13 or F-63 compounds could be translated in vivo, they have beentested in the MCF-7 xenograft model.

All animal procedures were performed according to the guidelines of the2010/63/UE Directive on the protection of animals used for scientificpurposes. The protocol was approved by the Animal Ethical Committee ofthe Pierre Fabre Institute. Five millions MCF-7 cells were injectedsubcutaneous into 7 weeks old Swiss/Nude mice. Prior to cell injection,oestrogen pellets (Innovative Research of America) were implanted to theleft flank to mice in order to release estrogens necessary to the invivo growth of MCF-7 tumors.

Twenty days after MCF-7 cell implantation, when tumors reached anaverage size of 120-150 mm³, the animals were divided into groups of 5mice according to tumor size and aspect. The different treatments wereinoculated by intraperitoneal injections. The health status of animalswas monitored daily. Tumor volume was measured twice a week with anelectronic calliper until study end. Tumor volume is calculated with thefollowing formula: π/6×length×width×height. Toxicity was evaluatedfollowing the weight of animals three times per week. Statisticalanalyses were performed at each measure using a Mann-Whitney test. Allcompounds were injected intraperitoneally (i.p.). In this example, theanti-tumor activity of c208F2 mAb coupled with either E-13, F-13 or F-63at about DAR 4 was evaluated after 2 injections of a 7 mg/kg dose at D20and D27 (FIGS. 22A, 22B and 22C). In parallel the capped-drug moietiesE-13, F-13 and F-63 were injected at the equivalent dose of the onecorresponding to 7 mg/kg of c208F2-E-13, c208F2-F-13 and c208F2-F-63 DARabout 4.

Injection of either c208-E-13 (FIG. 22A), c208F2-G-13 (FIG. 22B) orc208F2-F-63 (FIG. 22C) significantly inhibited and even induced acomplete tumor growth regression (p<0.05 vs corresponding capped-drug).No statistical activity difference between c208-E-13, c208F2-G-13 andc208F2-F-63 could be noted. Capped drugs had no effect on MCF-7 tumorgrowth (p >0.05 vs control group)

A second set of experiments was performed with c208F2 coupled witheither E-13 or G-13 and with the irrelevant antibody c9G4 coupled witheither E-13 or G-13 in MCF-7 xenograft models as described previously.Mice were injected i.p. with 7 mg/kg of each ADCs at D20 and D27 (FIGS.23A and 23B).

Injection of both c9G4-E-13 and c9G4-F-13 affected moderately andtransiently the growth of MCF-7 xenograft tumors. However, this secondexperiment confirmed that injections of either c208-E-13 or c208F2-G-13induced complete tumor regression since D43 showing the high anti-tumoractivity of those ADCs.

Example 25 Potent Cytotoxicity In Vitro of Axl ADCs Coupled withDifferent Drugs

The cytotoxic activity of ADCs for inhibition of tumor cell growth wastested in a cell proliferation assay using SN12C (Axl⁺ human renal cellcarcinoma) and MCF-7 (Axl⁻ human breast adenocarcinoma). Briefly, cellswere seeded into 96 well multi-well plates the day before drug treatmentat 2500 cells per well. ADCs and controls were serially diluted and thenadded to the mw-96 plates. Cells were then incubated for 6 days at 37°C. and 5% CO₂. The cell viability was quantified by measuring the levelof ATP in the wells using the CellTiter-Glo® Luminescent Cell ViabilityAssay (Promega catalog #G7571). The percentage of cell viability wascalculated considering untreated cells as 100%. Using a nonlinearregression analysis (GraphPad Prism 4.0), the IC₅₀, the concentration ofcompound needed to yield a 50% reduction in viability compared withuntreated cells (control=100%), was determined and expressed in molarity(FIG. 28).

Data showed in FIGS. 28A and 28B that hz1613F12 conjugated to eitherE-13 or G-13 give IC₅₀ values of 1.048 10⁻¹⁰ M, 1.413 10⁻¹⁰ M,respectively. Thus E-13 and G-13 can trigger a strong cytotocixity whencoupled to an Axl antibody such as 1613F12. No cytotoxicity is observedon MCF7 cells (Axl⁻).

Example 26 In Vivo Activity of the Trastuzumab Antibody Conjugated toEither E-13 or G-13 Compounds in the Calu-3 Xenograft Model

In order to confirm the in vivo efficacy of antibodies coupled to G-13or E-13 compounds, they have been coupled to Trastuzumab and tested inthe HER2 sensitive xenograft model Calu-3 known for its HER2amplification and (3⁺) expression. The antibody Tratuzumab was purchasedfrom Euromedex, 24 Rue des Tuileries 67460 SOUFFELWEYERSHEIM/France.

All animal procedures were performed according to the guidelines of the2010/63/UE Directive on the protection of animals used for scientificpurposes. The protocol was approved by the Animal Ethical Committee ofthe Pierre Fabre Institute. Seven millions Calu-3 cells were injectedsubcutaneous into 7 weeks old SCID mice.

Six days after Calu-3 cell implantation, when tumors reached an averagesize of 250-260 mm³, the animals were divided into groups of 6 miceaccording to tumor size and aspect. The different treatments wereinoculated by intraperitoneal injections. The health status of animalswas monitored daily. Tumor volume was measured twice a week with anelectronic calliper until study end. Tumor volume is calculated with thefollowing formula: (length×width²)/2. Toxicity was evaluated followingthe weight of animals three times per week. Statistical analyses wereperformed at each measure using a Mann-Whitney test. All compounds wereinjected intraperitoneally (i.p.). In this example, the anti-tumoractivity of Tratuzumab mAb coupled with either E-13 or G-13 at about DAR4 was evaluated after 1 injections of a 3 mg/kg dose at D6. In parallelTrastuzumab alone was injected at the equivalent dose of the onecorresponding to 3 mg/kg of naked antibody.

Injection of either Trastuzumab-E-13 (FIG. 29A) or Trastuzumab-G-13(FIG. 29B) significantly inhibited the tumor growth and even induced acomplete tumor growth regression in all treated mice (p<0.05 vscorresponding naked antibody). No statistical activity difference wasobserved between Trastuzumab-E-13, and Trastuzumab-G-13 groups. Comparedto published data (Cretella et al. Molecular Cancer 2014, 13:143) onthis Calu-3 model, TDM-1 did not induced complete regression even if itsdosing was higher (15 mg/kg every 6 days vs 3 mg/kg, one injectionrespectively) than the one used for either Trastuzumab-E-13 orTrastuzumab-G-13.

Example 27 In Vivo Activity of the Trastuzumab Antibody Conjugated toEither E-13 or G-13 Compounds in the JIMT-1 Xenograft Model

In order to know whether Trastuzumab antibody conjugates display also anactivity on a model known to be resistant to Trastuzumab, the JIMT-1xenograft model that highly expressed HER2 but that was resistant toTrastuzumab therapy was evaluated. All animal procedures were performedaccording to the guidelines of the 2010/63/UE Directive on theprotection of animals used for scientific purposes. The protocol wasapproved by the Animal Ethical Committee of the Pierre Fabre Institute.Seven millions JIMT-1 cells were injected subcutaneous into 7 weeks oldSCID mice.

Fourteen days after JIMT-1 cell implantation, when tumors reached anaverage size of 220-230 mm³, the animals were divided into groups of 5mice according to tumor size and aspect. The different treatments wereinoculated by intraperitoneal injections. The health status of animalswas monitored daily. Tumor volume was measured twice a week with anelectronic calliper until study end. Tumor volume is calculated with thefollowing formula: (length×width²)/2. Toxicity was evaluated followingthe weight of animals three times per week. Statistical analyses wereperformed at each measure using a Mann-Whitney test. All compounds wereinjected intraperitoneally (i.p.). In this example, the anti-tumoractivity of Trastuzumab mAb coupled with either E-13 or G-13 at aboutDAR 4 was evaluated after 1 injections of a 3 mg/kg dose at D6 (FIGS.30A and 30B). In a first experiment, we have showed that Trastuzumabalone did not have any anti-tumoral effect (FIG. 30C). This result is inagreement with published data.

Injection of either Trastuzumab-E-13 (FIG. 30A) or Trastuzumab-G-13(FIG. 30B) significantly inhibited the tumor growth traduced byrespectively 73 and 70% of growth inhibition at day 34. No statisticalactivity difference could be noted between Trastuzumab-E-13, andTrastuzumab-G-13. As already observed in the Calu-3 model and comparedto published data (Barok et al. Breast Cancer Research 2011, 13:R46) onthis JIMT-1 model, TDM-1 seems to be less potent even if its dosing (15mg/kg every 6 days vs 3 mg/kg, one injection respectively) was higherthan the one used for either Trastuzumab-E-13 or Trastuzumab-G-13.

The invention claimed is:
 1. An antibody-drug-conjugate of the followingformula (I):Ab-(L-D)_(n)  (I) or a pharmaceutically acceptable salt thereof,wherein: Ab is a target antibody or an antigen antibody, or a targetbinding fragment thereof or an antigen binding fragment thereof; L is alinker of the following formula (III):

wherein: L₂ is (C₄-C₁₀)cycloalkyl-carbonyl, (C₂-C₆)alkyl, or(C₂-C₆)alkyl-carbonyl; W is an amino acid; w is an integer between 0 and5; Y is p-aminobenzoyloxycarbonyl (PAB-carbonyl) with PAB being

y is 0 when w is 0 and y is 1 when w is an integer between 1 and 5; theasterisk indicates the point of attachment to D; the wavy line indicatesthe point of attachment to Ab; D is a drug moiety of the followingformula (II):

wherein: R₁ is H or OH; R₂ is (C₁-C₆)alkyl, COOH, COO-((C₁-C6)alkyl) orthiazolyl; R₃ is H or a (C₁-C₆)alkyl group; A is: a group of formula-Het-Alk-, wherein Alk is a (C₁-C₈) alkanediyl group and is linked toNR₃, and Het is a heterocycle optionally substituted by a (C₁-C₆) alkylgroup and containing at least one nitrogen atom, said nitrogen atombeing linked to L, or a group of formula -A_(a)-A_(b)-, wherein A_(a) islinked to L and is O or NR₉ with R₉ being H or (C₁-C6)alkyl, and A_(b)is linked to NR₃ and is: an aryl-(C₁-C8)alkanediyl orheterocycle-(C₁-C₈)alkanediyl group, said group being optionallysubstituted by a (C₁-C₆)alkyl group, the aryl or heterocycle moietybeing linked to A_(a) and the (C₁-C₈)alkanediyl moiety being linked toNR₃; the wavy line indicates the point of attachment to L; and n is aninteger between 1 and
 12. 2. The antibody-drug-conjugate of claim 1,wherein the target or antigen of said Ab or a target binding fragmentthereof or an antigen binding fragment thereof is selected from clusterof differentiation 19 (CD19), cluster of differentiation 20 (CD20),cluster of differentiation 22 (CD22), cluster of differentiation25(CD25), cluster of differentiation 30 (CD30), cluster ofdifferentiation 33 (CD33), cluster of differentiation 40 (CD40), clusterof differentiation 56 (CD56), cluster of differentiation 64 (CD64),cluster of differentiation 70 (CD70), cluster of differentiation 74(CD74), cluster of differentiation 79 (CD79), cluster of differentiation105 (CD105), cluster of differentiation 138 (CD138), cluster ofdifferentiation 174 (CD174), cluster of differentiation 205 (CD205),cluster of differentiation 227 (CD227), cluster of differentiation 326(CD326), cluster of differentiation 340 (CD340), mucin 16 (MUC16),glycoprotein Nmb (GPNMB), prostate specific membrane antigen (PSMA),cripto, extra-domain B (ED-B), transmembrane protein with epidermalgrowth factor like and two follistatin like domains 2 (TMEFF2), ephrintype-B receptor 2 (EphB2), ephrin type-A receptor 2 (EphA2), fibroblastactivation protein (FAP), alpha-V integrin (av integrin), mesothelin,epidermal growth factor receptor (EGFR), tumor-associated glycoprotein72 (TAG-72), disialoganglioside 2 (GD2), carbonic anhydrase 9 (CAIX),5T4, human epidermal growth factor receptor 1 (HER1), human epidermalgrowth factor receptor 3 (HER3), human epidermal growth factor receptor2 (HER2), insulin-like growth factor 1 (IGF-1R), Axl, and their extracellular membrane (ECD) fragment.
 3. The antibody-drug-conjugate ofclaim 1, wherein said target or antigen of said Ab or a target bindingfragment thereof or an antigen binding fragment thereof is selected fromthe group consisting of HER2, IGF-1R, Axl, and the extra cellularmembrane (ECD) fragment thereof.
 4. The antibody-drug-conjugate of claim1, wherein said Ab is an antibody, or an antigen binding fragmentthereof, and wherein said Ab binds to human IGF-1 R and is selected fromthe group consisting of: i) the antibodies 208F2, 212A11, 214F8, 219D6and 213B10; ii) the antibodies which compete for binding to IGF-1R withthe antibodies of i); and iii) the antibodies which bind to the sameepitope of IGF-1R as the antibodies of i).
 5. Theantibody-drug-conjugate of claim 1, wherein said Ab is an antibody, oran antigen binding fragment thereof, and wherein said Ab binds to humanIGF-1R and is selected from the group consisting of: i) an antibodywhich comprises the three heavy chain complementarity determiningregions (CDR_(s)) of sequence SEQ ID NOs: 1, 2 and 3 and the three lightchain CDRs of sequence SEQ ID NOs: 4, 5 and 6; ii) an antibody whichcompetes for binding to IGF-1R with the antibody of i); and iii) anantibody which binds to the same epitope of IGF-1R as the antibody ofi).
 6. The antibody-drug-conjugate of claim 1, wherein said Abcomprises: a) a heavy chain variable domain (VH) of sequence SEQ ID NO:33, wherein said sequence SEQ ID NO: 33 comprises at least 1back-mutation selected from the residues 20, 34, 35, 38, 48, 50, 59, 61,62, 70, 72, 74, 76, 77, 79, 82 or 95; and b) a light chain variabledomain (VL) of sequence SEQ ID NO: 35, wherein said sequence SEQ ID NO:35 comprises at least 1 back-mutation selected from the residues 22, 53,55, 65, 71, 72, 77 or
 87. 7. The antibody-drug-conjugate of claim 1,wherein said Ab is an antibody, or an antigen binding fragment thereof,and wherein said Ab binds to human protein Axl and is selected from thegroup consisting of: i) an antibody which comprises the three heavychain complementarity determining regions (CDRs) of sequence SEQ ID NOs:59, 60 and 61 and the three light chain CDRs of sequence SEQ ID NOs: 56,57 and 58; ii) an antibody which competes for binding to Axl with theantibody of i); and iii) an antibody which binds to the same epitope ofAxl as the antibody of i).
 8. The antibody-drug-conjugate of claim 1,wherein said Ab is an antibody or an antigen binding fragment thereof,and wherein said Ab binds to human HER2 and consists of trastuzumab. 9.The antibody-drug-conjugate of claim 1, wherein L₂is the followingformula:

and wherein: the asterisk indicates the point of attachment to (W)_(w);and the wavy line indicates the point of attachment to the nitrogen atomof the maleimide moiety.
 10. The antibody-drug-conjugate of claim 1,wherein w =0; or w =2 and (W)_(w) is selected from the group consistingof:

and wherein: the asterisk indicates the point of attachment to (Y)_(y);and the wavy line indicates the point of attachment to L₂.
 11. Theantibody-drug-conjugate of claim 1, wherein L is selected from the groupconsisting of:

and wherein the asterisk indicates the point of attachment to D, and thewavy line indicates the point of attachment to Ab.
 12. Theantibody-drug-conjugate of claim 1, wherein A is a group of formula-A_(a)-A_(b)-, and wherein A_(a) is as defined in claim 1 and A_(b) is:phenyl-(C₁-C₂)alkanediyl, or heterocycle-(C1-C2)alkanediyl optionallysubstituted by a (C1-C6)alkyl group, the heterocycle being a saturated,unsaturated or aromatic ring with 5 or 6 members comprising 1 or 2nitrogen atoms.
 13. The antibody-drug-conjugate of claim 1, wherein A isa group of the following formula:

wherein: R₉ is as defined in claim 1 and m is an integer between 1 and8, the wavy line indicates the point of attachment to L, and theasterisk indicates the point of attachment to NR₃.
 14. Theantibody-drug-conjugate of claim 1, wherein (L-D) is selected from thegroup consisting of:

and wherein the wavy line indicates the point of attachment to Ab. 15.The antibody-drug-conjugate according to claim 1, wherein theantibody-drug conjugate has a formula selected from the group consistingof:

or a pharmaceutically acceptable salt thereof, wherein Ab is a targetantibody or an antigen antibody, or a target binding fragment thereof oran antigen binding fragment thereof.
 16. The antibody-drug-conjugate ofclaim 15, wherein the target or antigen of said Ab or a target bindingfragment thereof or an antigen binding fragment thereof is selected fromthe group consisting of CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56,CD64, CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340,MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2, FAP, avintegrin, mesothelin, EGFR, TAG-72, GD2, CAIX, 5T4, HER1, HER3, HER2,IGF-1R, Axl and their extra cellular membrane (ECD) fragment.
 17. Theantibody-drug-conjugate of claim 15, wherein the target or antigen ofsaid Ab or a target binding fragment thereof or an antigen bindingfragment thereof is selected from the group consisting of HER2, IGF-1Rand Axl.
 18. The antibody-drug-conjugate of claim 15, wherein the targetor antigen of said Ab or a target binding fragment thereof or an antigenbinding fragment thereof is selected from the group consisting of humanHER2, human IGF-1R and human Axl.
 19. The antibody-drug-conjugate ofclaim 15, wherein said Ab is an antibody, or an antigen binding fragmentthereof, and is selected from the group consisting of: a) an Ab, or anantigen binding fragment thereof, wherein said Ab binds to human IGF-1Rand is selected from the group consisting of: i) the antibodies 208F2,212A11, 214F8, 219D6 and 213B10; ii) the antibodies which compete forbinding to IGF-1R with the antibodies of i); and iii) the antibodieswhich bind to the same epitope of IGF-1R as the antibodies of i), b) anAb, or an antigen binding fragment thereof, wherein said Ab binds tohuman IGF-1R and is selected from the group consisting of: i) anantibody which comprises the three heavy chain complementaritydetermining regions (CDRs) of sequence SEQ ID NOs: 1, 2 and 3 and thethree light chain CDRs of sequence SEQ ID NOs: 4, 5 and 6; ii) anantibody which competes for binding to IGF-1R with the antibody of i);and iii) an antibody which binds to the same epitope of IGF-1R as theantibody of i), c) an Ab which comprises: i) a heavy chain variabledomain (VH) of sequence SEQ ID NO: 33, wherein said sequence SEQ ID NO:33 comprises at least 1 back-mutation selected from the residues 20, 34,35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 or 95; and ii) alight chain variable domain (VL) of sequence SEQ ID NO: 35, wherein saidsequence SEQ ID NO: 35 comprises at least 1 back-mutation selected fromthe residues 22, 53, 55, 65, 71, 72, 77 or 87, d) an Ab, or an antigenbinding fragment thereof, wherein said Ab binds to human Axl and isselected from the group consisting of: i) an antibody which comprisesthe three heavy chain CDRs of sequence SEQ ID NOs: 59, 60 and 61 and thethree light chain complementarity determining regions (CDRs) of sequenceSEQ ID NOs: 56, 57 and 58; ii) an antibody which competes for binding toAxl with the antibody of i); and iii) an antibody which binds to thesame epitope of Axl as the antibody of i), and e) an Ab, or an antigenbinding fragment thereof, wherein said Ab binds to human HER2 consistingof trastuzumab or pertuzumab.
 20. The antibody-drug-conjugate of claim1, wherein n is
 2. 21. The antibody-drug-conjugate of claim 1, wherein nis
 4. 22. A composition comprising at least one antibody-drug-conjugateof claim
 1. 23. The composition of claim 22, further comprising apharmaceutically acceptable vehicle.